The uptake and metabolism of uniformly 14C-labeled adenosine and the effect of cordycepin (3′-deoxyadenosine) and its 5′-phosphates on Cordyceps militaris have been studied. The uptake of [ 14C]adenosine by C. militaris was more rapid than the subsequent in vivo phosphorylation, conversion to cordycepin and incorporation into RNA. The distribution of 14C in cordycepin, the acid-soluble pool and RNA of cordycepin-producing cultures of C. militaris 24 h after the administration of [ 14C]adenosine was 24, 18 and 15%, respectively. Radioactive adenine, adenosine, AMP, ADP and ATP were isolated from the acid-soluble pool of C. militaris. A very small amount of uniformly 14C-labeled cordycepin was found in the acid-soluble pool. The purine nucleotides, AMP, GMP, IMP and XMP were isolated from the hydrolyzed RNA. Cell-free extracts of C. militaris rapidly hydrolyzed the 5′-mono- and 5′-triphos phates of cordycepin. Cordycepin and cordycepin 5′-monophosphate were not deaminated by adenosine aminohydrolase or AMP aminohydrolase. The 40 000 × g supernatant from C. militaris phosphorylated adenosine, 2′-deoxyadenosine, the pyrrolopyrimidine nucleoside antibiotics and psicofuranine but not cordycepin, uridine or cytidine. Ribosephosphate pyrophosphokinase from C. militaris was strongly inhibited by cordycepin 5′-triphosphate. Cordycepin 5′-monophosphate inhibited 5-phosphoribosyl-1-pyrophosphate amidotransferase. These studies in vitro suggest that these same enzymes that are inhibited by cordycepin 5′-mono- and 5′-triphosphates in Bacillus subtilis, pigeon liver and Ehrlich ascites tumor cells are also inhibited in C. militaris. Uniformly 3H-labeled cordycepin was not taken up by C. militaris. A 3′-ribonucleotide reductase, similar to the 2′-ribonucleotide reductase of Lactobacillus leichmannii and Escherichia coli, could not be demonstrated with cell-free extracts of C. militaris with adenosine, AMP, ADP or ATP as substrates. 3-Deoxyribose 5-phosphate was not a substrate for the biosynthesis of cordycepin in vitro.