Rhodococcus-E. coli shuttle vector pNC9501 was constructed using circular cryptic plasmid pNC903 from propene-degrading Rhodococcus ruber P-II-123-1. Sequence analysis of pNC903 revealed two open-reading frames encoding the replication proteins Reps A and B. In the amino acid sequence of the putative Rep B, a helix-turn-helix motif, which is responsible for the binding of DNA, was found. Sequencing of the upstream region of the putative Rep A and incompatibility tests revealed that pNC903 is a Mycobacterium-derived pAL5000-related plasmid. pNC9501 could also be transformed into Mycobacterium sp. showing good segregation stability (< 0.1% plasmid loss/generation) in the absence of selective pressure.