In a recent publication, evidence was presented that cellular immune responses are associated with increased in vivo and in vitro excretion of neopterin. Our study aimed at investigating the biosynthesis of unconjugated pterins in highly purified human macrophages and T lymphocytes before and during stimulation with supernatants of activated T cells or with recombinant human interferon-gamma (IFN-gamma) by monitoring the following parameters: substrate concentration (GTP, guanosine triphosphate), activity of the enzyme initiating the biosynthesis of pterins (GTP-cyclohydrolase I) and product concentrations of total neopterin, biopterin, and pterin. In contrast to T cells and other tissues, macrophages were unable to produce tetrahydrobiopterin. This was indicated by our failure to detect biopterin and pterin. Instead, products of the first biosynthetic step accumulated, which were measured as total neopterin. We concluded that in macrophages the other enzymes required for biosynthesis of tetrahydrobiopterin are limiting. GTP concentration correlated with GTP cyclohydrolase I activity. An increase in both was induced by IFN-gamma and suppressed by neutralization of T-cell supernatants with monoclonal antibodies having specificity for IFN-gamma. Addition of tetrahydrobiopterin to the culture medium only led to a suppressed increase in GTP cyclohydrolase I activity and neopterin, but not in GTP concentration. Thus, it appears that IFN-gamma selectively stimulates the early steps of pterin biosynthesis in macrophages, thereby leading to accumulation and excretion of dihydroneopterin and neopterin. Although the physiological role of this phenomenon remains obscure, the fact that it seems to reflect endogenous release of IFN-gamma deserves particular attention.