Studies with purified enzymes have shown that 2'-deoxycoformycin (dCF) is a potent and selective inhibitor of adenosine deaminase (ADA). Specificity of dCF's effects on adenosine metabolism in intact human skin fibroblasts was investigated by examining the isotopic flux from exogenous [14C] adenosine to metabolic products in hypoxanthine phosphoribosyltransferase deficient (HPRT-) cells which cannot recycle hypoxanthine. Apparent ADA activity (as estimated by isotopic flux to inosine and hypoxanthine) was profoundly inhibited by dCF (with at least 50% inhibition at 10(-8) M and 95% inhibition at 10(-5) M dCF). The degree of inhibition was similar at various exogenous adenosine concentrations ranging from 1 to 400 microM. Some inhibition of isotopic flux to adenine nucleotides (an ADA independent process in HPRT- cells) could be demonstrated, but only in media containing high concentrations of adenosine. Even at 400 microM adenosine, the highest concentration employed, isotopic flux to adenine nucleotides was unaffected by concentrations of dCF below 10(-6) M, and only 30% inhibition was achieved with 10(-5) M dCF. Inhibition of adenosine phosphorylation to AMP appears to be the most likely explanation for dCF inhibition of isotopic flux from [14C] adenosine to adenine nucleotides, probably due to substrate inhibition of adenosine kinase by high levels of intracellular adenosine produced when ADA is inhibited by dCF. No evidence for dCF inhibition of either adenosine transport or phosphorylations within the adenine nucleotide pool (from AMP to ADP or from ADP to ATP) was found. Thus, at physiological levels of exogenous adenosine (0.03 to 2.6 microM), dCF appears to be a potent and highly specific inhibitor of ADA in human skin fibroblasts.