The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame for a 33-amino-acid toxin called Fst. Expression of fst is regulated post-transcriptionally by RNA II. In this paper, RNA II is shown to interact with RNA I by a unique antisense RNA mechanism. RNA I and RNA II contain complementary direct repeats at their 5' ends and a complementary transcriptional terminator stem-loop at their 3' ends. Deletion of the terminator or mutations within the terminator loop of RNA II severely reduced the rate of interaction in vitro. Mutations in the 5' direct repeats of RNA II prevented the RNAs from interacting in vitro. For these mutations in RNA II, complementary mutations in RNA I were shown to restore interaction. The reduced binding efficiency of the RNA II mutants was paralleled by the failure of these mutants to suppress par-mediated killing in vivo. These results indicate that regions at both the 5' and the 3' ends of the par transcripts are important for RNA I-RNA II interaction.