N-Acetyltransferase (NAT) activity towards p-aminobenzoic acid and sulfamethazine was examined in primary cultures of rat hepatocytes cultured on three extracellular matrices (ECM)-type I collagen, thermally denatured type I collagen, and Matrigel((R)). Whereas protein and DNA content declined markedly during the first 24 hr of culture, p-acetylamidobenzoate (AcPABA) and N-acetylsulfamethazine (AcSMZ) formation were readily detectable on all three ECM for the 6-day culture period. Protein and DNA content, as well as NAT activities, were higher on Matrigel than on either of the other two ECM. Additional studies were conducted to confirm the expression of both enzymes during the culture period. The ratio of AcPABA to AcSMZ formation remained relatively stable throughout the 6-day culture period, suggesting that both enzymes continued to be expressed throughout the study period. Further studies in cells cultured on Matrigel revealed that AcPABA formation exhibited a time-dependent decline when cytosol from cultured cells was incubated at 50 degrees C, whereas AcSMZ formation proved to be thermostable. Moreover, methotrexate substantially inhibited AcPABA formation, but had only modest effects on AcSMZ. These studies support the conclusion that AcPABA and AcSMZ are predominantly formed by way of different enzymes throughout the culture period. These findings are supported by the observation that NAT1 and NAT2 mRNA were detectable on all days examined. These data indicate that primary cultures of rat hepatocytes should prove useful in probing the regulation of NAT and its role in toxicity.