Reverse transcriptases (RT) are found in a minor population of Escherichia coli and are responsible for the synthesis of multicopy single-stranded DNA. These RTs specifically recognize RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). Here, we purified the 66-residue, C-terminal fragment of RT-Ec86, RT from E. coli, which is responsible for the synthesis of multicopy single-stranded DNA-Ec86. This fragment, RT-Ec86-(255-320), was found to consist mainly of alpha-helical structures on the basis of its CD spectrum, which is consistent with the prediction of this region as the thumb domain from the structural alignment of RT-Ec86 with human immunodeficiency virus-1 RT. RT-Ec86-(255-320) was able to bind to a 28-base synthetic RNA consisting of the 5'-end single-stranded RNA containing the branching G residue and the recognition stem-loop structure in the RT-Ec86 primer-template RNA with a Kd value of 5 x 10(-8) M. By stepwise shortening of the 5'-end single-stranded region of the RNA, RT-Ec86-(255-320) was found still to be able to form a stable complex with only the stem-loop structure consisting of an 8-bp stem and a 3-base loop. In this stem-loop structure, the UUU loop was essential for the complex formation. RT-Ec73-(251-316) from another E. coli RT could not bind to the 28-base RNA for RT-Ec86 but could bind to its own stem-loop structure having a 3-base AGU loop. These results support the notion that the highly diverse C-terminal regions of bacterial RTs play an important role in recognizing their own specific primer-template RNA structure for the cDNA priming reaction.