A restriction map of Mycobacterium fortuitum var. fortuitum plasmid pAL5000 was established. The unique sites for ApaI, BamHI, BglII, BstEII, ClaI, EcoRI, EcoRV, HpaI, KpnI and NarI were located on the 5.0-Kb plasmid. The plasmid had no sites for AhaIII, BclI, HindIII, PstI, SphI and XbaI. pAL5000 was cloned into pBR322 and propagated in Escherichia coli. Three hybrid pAL5000-pBR322 plasmids carrying the complete pAL5000 sequence were constructed by joining the plasmids at their BamHI, EcoRI or EcoRV sites. We also cloned into these plasmids a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The construction of these plasmids will facilitate the analysis and manipulation of pAL5000, and may allow the development of a vector system for genetic analysis in mycobacteria.