Bacteriophage SPO1 DNA-negative (D0) mutants were tested for the induction of viral DNA polymerase during Bacillus subtilis infection. Extracts from SPO1-infected bacteria exhibited enzymatic activity when representative mutants of seven out of the nine known D0 genes were employed. This activity was undetectable in cells infected with mutants in genes 28 and 31. The product of gene 28 (gp28) is known to be responsible for turning on SPO1 middle gene expression. Results show that nonsense mutation in gene 31 leads to the absence of a single polypeptide of 100-105 kDa and that phage DNA synthesis "in vivo" directly depends on gp31 activity. Based on these data it is proposed that SPO1 gene 31 codes for the viral DNA polymerase.