Mammalian genes encoding a 35-kDa peroxisomal membrane protein (PMP35, peroxisome assembly factor-1) are compared using the polymerase chain reaction and DNA sequencing. DNA sequencing of the 915 bp of the PMP35 coding regions was in complete agreement with previously published rat data and showed 36 and 133 nucleotide substitutions, respectively, in mouse and man. The 12 and 35 respective amino acid changes encoded by these nucleotide substitutions are clustered and compatible with putative membrane-spanning regions. Rat/human and rat/mouse comparisons yield silent mutation rates of 0.33 and 0.21% per site per million years and replacement mutation rates of 0.082 and 0.076%; transitions account for 67% (human/mouse) and 83% (rat/mouse) of nucleotide replacements among PMP35 genes. PMP35 gene expression in mouse tissues as measured by reverse transcriptase-PCR was responsive to clofibrate and disproportionately high in neural tissue.