Aminooligopeptidase (AOP) is the predominant peptidase in the small intestinal epithelium. During postnatal development in the rat, characteristic ontogenic and regional patterns of AOP activities become established. To investigate the molecular mechanisms regulating the maturational changes in AOP activity, we compared AOP synthesis and assembly in the jejunum of suckling (14-day-old) and weaned (28-day-old) rats. AOP synthesis was assessed in vivo by intraperitoneal labeling with [35S]methionine. Both AOP activity and AOP synthesis doubled at weaning, while its posttranslational processing remained unaltered. To examine the mechanisms responsible for generating the regional differences in AOP activity in weaned rats, we contrasted the de novo synthesis, kinetic properties, total immunoreactive protein, and degradation of the jejunal and ileal peptidases. Although AOP catalytic activity was significantly greater in the jejunum than in the ileum, its synthesis rate and substrate affinity (Km) were identical at both sites. However, the ileal peptidase was degraded more rapidly than the jejunal enzyme (t1/2 = 4 and 10 h, respectively). In summary, our data show that increased synthesis accounts for the ontogenic rise in intestinal AOP activity but that altered enzyme turnover produces the jejunal-ileal gradient in AOP activity in weaned rats. Thus the ontogenic and regional expressions of intestinal AOP are defined by transcriptional/translational and posttranscriptional regulatory mechanisms, respectively.