The nucleotide sequence of the 1847-bp Bifidobacterium longum B2577 cryptic plasmid pMB1 was determined. The plasmid had a G+C content of 62.0%, and contained two open reading frames, orf1 and orf2, likely arranged in an operon. The proteins encoded by orf1 and orf2 show the highest degree of similarity with similarly arranged peptide sequences translated from Corynebacterium glutamicum pXZ10142 and Mycobacterium fortuitum pAL5000 plasmids. Recombinant plasmids containing the pMB1 replicon were able to replicate in Bifidobacterium animalis MB209. The successful transformation of this strain with pMB1-based plasmids facilitated characterization of this replicon, results of which showed that both orf1 and orf2 are necessary for plasmid replication. A family of new Escherichia coli-B. animalis shuttle plasmids, based on the pMB1 replicon and expressing a cat and an ery gene, was constructed.