The rumen ecosystem represents a classic example of a mutualistic relationship between host animals and microbial populations. Urea nitrogen secreted from the blood to the rumen is a crucial factor shaping this relationship. The transport of urea across rumen epithelium is facilitated by urea channels, namely UT-B and possibly AQP3. However, how ruminants regulate nitrogen availability to microbes through these transport proteins remain unclear. Given that calves are the stage at which of rumen microbe colonization begins, the developing rumen is an excellent model to observe this regulation. This study investigated the localization of urea channels (UT-B and AQP3) during rumen development. Using rumen epithelium samples of calves aged from birth to three months old (0, 7, 14, 21, 28, and 96 days old; weaned at 56 days old; n = 4-8 at each age), immunolocalization of UT-B and AQP3 protein was performed. Initial results showed that, although abundance varied between the individual animals within each age groups, UT-B protein was found expressed at all six stages (n = 9/14 ). The immunostaining started from the cells of stratum basale at all stages (n = 9/14) and progressively extended to the adjacent cell layers toward the rumen lumen as the rumen papillae developed (n = 3/14). The strongest staining appeared to be found in stratum basale cells (n = 9/14), especially those surrounding large blood vessels (n = 3/14). The staining in stratum spinosum was mainly present in well-developed papillae at day 96 and was weak (n = 3/4), while stratum granulosum and stratum corneum were mainly negative throughout the six stages (n = 12/14). Occasionally, strong staining was found in balloon cells of stratum corneum at age day 96 (n = 2/4). The subcellular staining was clearly seen in the cells of stratum basale at the age of day 96, with the strongest density at the basolateral side of plasma membrane facing blood vessels (n = 3/4). In contrast to UT-B, the staining of AQP3 was only found in cells of stratum corneum at stages of day 28 (n=1/2) and day 96 (n = 2/3). Overall, the localization pattern of UT-B is consistent with the hypothesised functional organization of transporting urea nitrogen from blood to rumen. The localization of AQP3 in stratum corneum is unexpected and the physiological meaning is unclear. In on-going studies, the staining of UT-B and AQP3 will be quantified between the six stages. Given that papillae development is a key reflection of rumen function, precise measurement of papillae development will be also undertaken. Overall, these results give insight into how ruminant host animals regulate nitrogen availability, and hence commensal microbial populations, through altering the permeability of the rumen. Keywords: developing rumen, UT-B, AQP3, calves