ABSTRACT The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB ⁺ plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA ⁺ plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA ⁺ plasmid but differed over an ≈22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes ( vapB and vapJ to - M , with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA ⁺ plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.