Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and structurally related compounds. We cloned mouse liver NNMT cDNA to make it possible to test the hypothesis that large differences among strains in levels of hepatic NNMT activity might be associated with strain-dependent variation in NNMT amino acid sequence. Mouse liver NNMT cDNA was 1015 nucleotides in length with a 792 nucleotide open reading frame (ORF) that was 83% identical to the nucleotide sequence of the human liver NNMT cDNA ORF. The mouse liver cDNA encoded a 264 amino acid protein with a calculated Mr value of 29.6 kDa. NNMT cDNA ORF sequences were then determined in five inbred strains of mice with very different levels of hepatic NNMT enzymatic activity. Although multiple differences among strains in nucleotide sequence were observed, none altered encoded amino acids. cDNA sequences for C57BL/6J and C3H/HeJ mice, prototypic strains with "high" and "low" levels of hepatic NNMT activity, respectively, were then expressed in COS-1 cells. Both expression constructs yielded comparable levels of enzyme activity, and biochemical properties of the expressed enzyme, including apparent Km values for substrates and IC50 values for inhibition by N1-methylnicotinamide, were very similar to those of mouse liver NNMT. Growth and development experiments were then conducted, which demonstrated that, although at 8 weeks of age average hepatic NNMT activity in C57BL/6J mice was 5-fold higher than that in C3H/HeJ mice, activities in the two strains were comparable by 30 weeks of age--indicating strain-dependent variation in the developmental expression of NNMT in mouse liver. These observations will serve to focus future studies of strain-dependent differences in murine hepatic NNMT on the regulation of the enzyme activity during growth and development.