Acrylamide gel electrophoresis of mouse urine distinguishes two groups of pepsinogens, the fast-migrating group A and the slow-migrating group B. Inbred mouse strains fall into two categories with respect to group B pepsinogens, one expressing a single-banded and the other a double-banded phenotype. This variation is controlled by a single locus, urinary pepsinogen-2 or Upg-2, which has at least two alleles, Upg-2s and Upg-2d. Typing of recombinant inbred strains suggests that the location of the Upg-2 locus is on chromosome 1, in the vicinity of loci coding for other peptidases, namely, Pep-2 and Rnr, and of the Acf-1 locus.