Folate catabolism involves cleavage of the C(9)-N(10) bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.