We report mutations affecting several steps of CoIE1 primer formation. Primer precursor forms the hybrid with the template DNA and is cleaved by RNAase H to form the primer. Point mutations at positions -264, -265, -268, and -308 (base pairs upstream of the replication origin) reduce efficiency of hybrid formation. A suppressor mutation at -18 increases the efficiency for a mutant transcript and reduces that for a wild-type transcript. Therefore, hybrid formation probably starts after transcription passes this position. A mutation at -10 affects the site of cleavage by RNAase H. The site appears to be determined by the distance from a stem-loop structure immediately upstream. A double mutation at -186 and -188 results in formation of an RNA secondary structure that prevents use of an RNAase H-cleaved transcript as primer.