Three DNA polymerase activities, one related to DNA pol III, have been extracted from a DNA membrane complex purified from Streptococcus pneumoniae. DNA pol III was purified 3300-fold, DNA pol II 2800-fold and DNA pol I 1800-fold. Based on inhibition analysis with a drug known to inhibit DNA pol III activity in Gram positive organisms. 6(p-hydroxyphenyl azo) uracil (HpU), 55% of the total DNA polymerase activity is represented by pol III. In contrast, only 3-5% of the total DNA polymerase activity is inhibited by HpU in crude extracts. The purification of the DNA membrane complex from pneumococcus is modified from an earlier procedure (Firshein 1972). The modified procedure results in the separation of three distinct DNA-protein-phospholipid subcomplexes of which the one described above contains most of the radioactivity derived from cells pulsed for a short time with (3H)-thymidine. Proteins are involved in binding DNA in each complex and the conformation of DNA in each complex may be different. All of the subcomplexes contain DNA polymerase activity partially sensitive to HpU. These results provide direct evidence for the structural integrity of a complex that may be involved in DNA replication in vivo.