Major urinary protein (MUP) genes were isolated from C57 genomic libraries, characterized by restriction enzyme mapping and compared with MUP genes isolated from BALB/c genomic libraries (Clark et al, 1982; Bishop et al, 1982). The conclusions drawn from the characterization of this new set of MUP genes are in agreement with those previously drawn from studies on the BALB/c MUP genes. Most MUP genes were found to share extensive homology in their transcription units and 5' and 3' flanking regions. Exceptions were those genes whose coding regions have been interupted by insertions and/or deletions. The MUP genes fall into two main groups based on hybridization criteria: group 1 and group 2 (Bishop et al, 1982). With the exception of one group 2 gene (BL-25/CL-2), restriction site homology was found to be greater within groups than between than. Restriction site homologies further divided the group 1 genes into two sub-groups. Sequence data revealed that the two sub-groups have different forms of an A-rich region located M0bp upstream of the TATA box. Messenger RNA from tissues that express MUP was shown to be more homologous to group 1 coding sequences than to group 2 coding sequences. In the liver, two forms of MUP mRNA can be distinguished. Group 1 sequences hybridized preferentially to the abundantly transcribed long form of the mRNA, while group 2 sequences hybridized preferentially to the short and rarer form of the mRNA. Genomic digests illustrated that two types of variation are found between the MUP genes of BALB/c and C57BL/Fa mice. The first relates to the presence of variant restriction fragments. Two cloned MUP genes carrying such fragments were identified. The second relates to variation in the intensity of common restriction fragments. Differences between the strains in the total number of MUP genes were not observed. Variation in the intensity of common restriction fragments are proposed to be the result of different homogenization events that took place in the mouse lineages from which BALB/c and C57BL/Fa were derived.