A small cryptic plasmid originating from Bifidobacterium breve NCFB 2258 was cloned and its complete nucleotide sequence determined, pCIBb1 is a circular DNA molecule, 5750 bp in size with a GC composition of 57%. Computer-assisted analysis identified 10 possible open reading frames (ORFs), seven of which could be assigned no function from homology searches. One ORF, rep (380 amino acids), was postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. Demonstration of single-stranded forms of the plasmid in cell lysates that could be specifically degraded by S1 nuclease provided experimental evidence to substantiate a replication mechanism via single-stranded intermediates. Two other ORFs, par. (199 amino acids) and an ftsK-like gene (286 amino acids), were assigned putative functions based on the presence of conserved motifs in their deduced proteins. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.