Progressive liver fibrosis in rats develops when they are fed a diet deficient in choline. This diet also results in a pronounced and selective decrease in the liver microsomal content of a phase I drug—metabolizing enzyme belonging to the cytochrome P-450III gene family. Because P-450III cytochromes characteristically catalyze the N-demethylation of erythromycin, we believed that the production of breath CO2 from erythromycin would be dramatically reduced in choline-deficient rats. However, when 12 choline-deficient rats were compared with 9 control rats, the reduction in CO2 production from erythromycin (mean decrease 71%) was essentially identical to that from aminopyrine (mean decrease 69%), a substrate believed to be metabolized normally by the hepatocyte in fibrotic liver disease. Furthermore, we found that the relative erythromycin and aminopyrine demethylase activities were comparable when measured in vitro in liver microsomes prepared from the choline-deficient rats. To determine the molecular basis for the erythromycin demethylase activity in the choline-deficient rats, the liver microsomes were subjected to immunoblot analysis using a variety of polyclonal and monoclonal antibodies capable of distinguishing individual P-450III—related proteins. Our studies confirm that a major erythromycin demethylase belonging to the P-450III family, termed P-450p, was greatly reduced in the choline-deficient rat liver. However, the specific concentration of a second P-450p—related protein was essentially normal and that of a third P-450p—related protein was actually increased in the choline-deficient rat liver. These changes occurred over weeks and months on the choline-deficient diet and are not consistent with “feminization” of the liver. The P-450p—related proteins appeared to catalyze erythromycin demethylase activity because antibodies recognizing them inhibited the majority (62%) of this activity in choline-deficient rat microsomes. Finally, RNA extracted from choline-deficient rat livers was hybridized on Northern blots with synthetic oligonucleotide probes that identify the only two known rat P-450III cDNAs, PCN-1 and PCN-2. Prolonged choline deficiency appeared to have no consistent effects on expression of these RNA species. We conclude that despite the down-regulation of a major erythromycin demethylase, this catalytic activity is relatively preserved in the choline-deficient rat, at least in part because of differential regulation of P-450III cytochromes. Erythromycin therefore appears to offer little advantage over aminopyrine as a substrate for assessing liver function in this model of chronic liver disease. (HEPATOLOGY 1990;12:1371–1378).