A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.