Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAMbeta1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the antisense system of plasmid pT181. The secondary structures of antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single-round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 antisense RNA was sufficient for stable complex formation, both stem-loop structures of pT181-RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30-50% in the absence of antisense RNA), the sense RNA of pT181 required antisense RNA for induced termination. Rate constants of formation of pT181 sense-antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10-fold faster than stable binding.