Extracellular nucleotides activate the P2 family of receptors, of which there are two subgroups. P2X receptors are ligand-gated ion channels while P2Y receptors are G protein-coupled. Activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, the P2Y₂ receptor is expressed in the proliferative basal layer (Dixon et al., 1999), the P2X₅ receptor is present in the differentiated layers, and the P2X₇ receptor is present in the apoptotic squamous layer (Grieg et al., 2003). In this study, we have investigated the source of extracellular nucleotides in a human keratinocyte cell line (HaCaT). We have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors and confocal microscopy to show that HaCaT cells release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in cell surface ATP-generation. HaCaT cells were seeded at a density of 1 x 10⁵ cells/well in 12 well plates and grown to confluence overnight and serum-starved for 1 hour prior to treatment. Values from controls (addition of treatments to medium alone) were subtracted from those in the presence of cells. Mean basal release of ATP was ≈100 nM/10⁶ cells/hour and was significantly higher than that of SaOS-2 cells (an osteoblast cell line; p<0.0001, students’ t-test, n=8). Addition of 10 μM ADP, to culture medium resulted in a 6-fold elevation of the ATP concentration. This effect occurs immediately and interestingly, ATP-generation and degradation appear to be balanced since the concentration of ATP remained stable for 90 minutes after addition (n=4). Addition of 10 μM GTP or UTP each resulted in a 3-fold increase in the ATP concentration, whilst 10 μM 2MeSADP, UDP or ATP had no effect (n=4). ADP to ATP conversion was inhibited by 10-300 μM diadenosine pentaphosphate, 12-48 μM oligomycin or 10 mM UDP, suggesting the involvement of cell surface adenylate kinase, F₁F₀ ATP synthase and nucleoside diphosphokinase (NDPK) respectively (n=4). These findings were supported by immunohistochemical localisation. Simultaneous addition of 10 μM ADP and 20 μM GTP elevated ATP above that for each nucleotide alone indicating that GTP is acting as a phosphate donor in the reaction N₁TP + N₂DP ↔ N₁DP + N₂TP (n=4). However, the activity of NDPK, F₁F₀ ATP synthase or the forward reaction of adenylate kinase does not fully account for the ATP content of the culture medium. We postulate that this is due to the reverse reaction of adenylate kinase (AMP + GTP ↔ ADP + GDP). We have previously shown that HaCaT cells express multiple P2 receptors (Burrell et al., 2003). In this study we now identify the potential sources of ATP required to activate these receptors and thus provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.