The gastrointestinal epithelia secrete and absorb large amounts of solvents and fluid, while being exposed to harsh conditions, such as very low pH, high CO2 tensions, food products, detergents, bacteria and their products, as well as noxious agents released by the immune cells of the organism itself. Protective properties of the gastrointestinal mucus layer have been postulated more than a decade ago, but it was clear to investigators that the mucus layer had to be traversed from the epithelium to the lumen by the very same products that it needed to be protected from, such as the gastric acid. What was actually going on directly at the epithelial surface? Researchers from Europe, Japan and the US pioneered the study of gastrointestinal surface pH measurements more than 3 decades ago, by advancing pH- and voltage-sensitive dual-barreled electrodes through the mucus layer of chambered isolated mucosa, and later also through the mucus of exteriorized vascularly perfused stomach of live rodents. The small intestine was probed in a similar fashion. The disadvantage was that the advancement of the microelectrode potentially disturbed the unstirred layer within the mucus. This problem was overcome two decades later, when the use of pH-sensitive fluorescent dyes and multiphoton imaging allowed dynamica monitoring of the pH immediately adjacent to the surface of the gastric mucosa. When adapting this method to other parts of the gastrointestinal tract, other pH-sensitive dyes need to be used, because of the rather small pH range within which these dyes change their emission intensities proportionally to changes in pH. Using the above methods, it became clear that pH- gradients formed within the unstirred fluid layer in the mucus between the acidic lumen and the neutral-slightly alkaline oH of the epithelial cell layer in the upper segments of the GI tract. In the jejunum, the surface microclimate was acidic compared to the neutral lumen and enterocyte pH values. What factors allow the buildup and stability of such pH-gradients? In order to understand the regulation of the surface microclimate in a segment of the gastrointestinal tract, it has to be amenable to experimental manipulation. For the study of gastric pH gradients, acid secretion was easy to stimulate or inhibit in rodents. It was found that the stimulation of acid secretion enhanced the alkalinity of the surface pH above the mucous surface cells, possibly caused by the “alkaline tide”, the HCO3- that is exported via the parietal cell basolateral membrane and enters the capillaries that bring blood to the surface regions of the gastric epithelium. However, the opposite, the stimulation and inhibition of HCO3- secretion, was far more difficult to achieve. Thus, gene-manipulated mice were used that lacked either important transporters or important regulatory molecules for HCO3- secretion. In the stomach, this allowed the dynamic assessment of the alkaline halo adjacent to a microscopic wound and its role in protecting the wounded epithelium from acid damage. In the jejunum, changes in epithelial surface pH were found to accompany and modify the nutrient and electrolyte absorptive processes. In the colon, alterations in surface pH, brought about by deletion of the major apical Cl-/HCO3- exchanger Slc26a3 (DRA), are associated with changes in the mucus layer quality, an altered microbiome composition, an increase susceptibility to damage, and even slight inflammatory changes in the nonchallenged colonic mucosa. A combination of fluorescent markers, applied as dyes, fluorescent bacteria, or genetically expressed in the epithelial layer, allows the simultaneous assessment of tight junction integrity, mucus layer permeability, as well as of some physiological states of the cell layer below. In summary, advances in electrophysiological and imaging techniques have allowed to study dynamic changes of the surface pH microclimate during physiological digestive processes and pathological derangements. The use of transgenic animals allows, to some extent, to tentatively establish causal relationships. The development of new fluorescent markers and transgenic animal models, as well as a combination of optical and electrophysiological techniques, will likely allow an simultaneous assessment of more qualities of the surface microclimate and the underlying mucosa.