The effects of abasic (AP) sites on RNA synthesis were studied in vitro, using T7 RNA polymerase and a plasmid template containing a T7 promoter. The presence of increasing numbers of AP sites caused a progressive decline in RNA synthesis. The average RNA chain length, calculated from the ratio of initiation to chain elongation, decreased with increasing numbers of AP sites, revealing that complete blocks must occur during synthesis. The probability that RNA polymerase would be blocked at an AP site in the DNA template strand was estimated to be 0.3 in our experimental conditions. These results demonstrate that RNA synthesis by T7 RNA polymerase is inhibited by AP sites and that readthrough of the lesion occurs more frequently than premature chain termination. Chemical reduction of AP sites in the template did not change the block/bypass pattern.