DNA sequence analysis of a 1.55-kb region located 10 kb from the left end of the orf virus NZ-2 strain (OV NZ2) genome revealed an open reading frame, B2L, encoding a protein with a predicted molecular weight of 41.67 kDa. This protein (p42K) shows 42% amino acid sequence identity to the vaccinia virus (VAC) major envelope antigen p37K. In addition, p42K shows homology to a protein encoded by molluscum contagiosum virus (42.8% identity) and another encoded by fowlpox virus (38.3% identity). These proteins are themselves homologues of the VAC p37K. B2L is actively transcribed after the onset of DNA replication and S1 nuclease analysis mapped the 5' end of the transcript to within the sequence TAAATG. A VAC recombinant capable of expressing the p42K gene was constructed and used as an antigen in radioimmune precipitations and lymphocyte transformation assays. These assays demonstrated that OV p42K is one of a limited number of OV proteins to which sheep mount a strong antibody response and which stimulate lymphocytes derived from draining lymph nodes following a natural infection with OV NZ2.