A total of 530 strains of rumen bacteria were screened for the presence of plasmid DNA. The percentage of plasmid-bearing strains was found to be the highest among theBacteroides/Prevotellagroup (9.9%), while it was less than 1% in theButyrivibrio(0.2%) andClostridium(0.6%) genera. A small cryptic plasmid pRAM4 fromPrevotella ruminicolaT31 was subcloned inEscherichia coliand completely sequenced. Two open reading frames, encoding potential polypeptides ofMr32,322 (ORF1) and 32,122 (ORF2) with limited sequence similarity to replication initiation and mobilization proteins, respectively, could be identified within the sequence. The region upstream from ORF1 had an AT-rich (75%) region followed by four 22-bp direct repeats, a structure characteristic of replication origins. The plasmid hybridized at high stringency with plasmids fromBacteroides/PrevotellaandButyrivibrio,and with pBR322, suggesting that at least regions of the plasmid are widespread.