Although the Pr65gag precursor polyproteins of Moloney murine leukaemia virus (M-MuLV) and of Rauscher murine leukaemia virus (R-MuLV) have the same apparent mol. wt. by SDS--polyacrylamide gel electrophoresis (SDS--PAGE), their initial approximately 40 000 dalton intermediate cleavage products differ in mol. wt., i.e. the M-MuLV product (Pr41.5gag) is 1500 daltons larger than the R-MuLV product (Pr40gag). We took advantage of this difference to show that in vitro cleavage of R-MuLV Pr65gag by the M-MuLV proteolytic activity gives rise to R-MuLV Pr40gag and not M-MuLV Pr41.5gag. This result suggests that the specificity for cleavage of the MuLV Pr65gag is built into the substrate.