The effects of nucleotide analogues on DNA synthesis were studied in nucleotide-permeable Escherichia coli cells. 2′,3′-Dideoxyribosylthymine 5′-triphosphate inhibited replicative DNA synthesis far more strongly than endonuclease-induced DNA repair synthesis. In φX174-infected cells this analogue caused the formation of short pieces (7 S) of φX replicative form DNA in which newly synthesized DNA was found covalently linked to preformed primer. Deoxyuridine triphosphate led to even shorter pieces (3 S) of φX replicative form DNA. Nicotinamide mononucleotide, an inhibitor of E. coli DNA ligase, promoted accumulation of short (10 S) E. coli DNA pieces. Newly synthesized 5′-triphosphate ends were not detected in these nucleotide-permeable cells during the early stages of φX replicative form replication, nor was evidence obtained that nucleoside monophosphates are immediate precursors of DNA.