Caffeine inhibits the activity of DNA polymerase I (E. coli) and its proteolytic large fragment in in vitro DNA replication system. DNA polymerase from Micrococcus luteus is also equally inhibited by caffeine. The extent of inhibition was more with the activated adenovirus, T4 and calf thymus DNA than with synthetic DNA template-primers. Results obtained from time-course studies indicated that caffeine inhibition reached maximum by 30 min of incubation. Enzyme kinetic studies showed that inhibition was competitive with respect to DNA template.