We have studied the interactions of chlorogenic acid (CHL) and 2-hydroxy-5-nitrobenzaldehyde (HNB) with the components of the rat hepatic glucose 6-phosphatase (Glc-6-Pase) system. CHL and HNB are competitive inhibitors of glucose 6-phosphate (Glc-6-P) hydrolysis in intact microsomes with Ki values of 0.26 and 0.22 mm, respectively. CHL is without effect on the enzyme of fully disrupted microsomes or the system inorganic pyrophosphatase (PPiase) activity. HNB is a potent competitive inhibitor of the system PPiase activity (Ki = 0.56 mm) and a somewhat weaker noncompetitive inhibitor of enzyme activity (Ki = 2.1 mm). These findings indicate CHL binds to T1, the Glc-6-P transporter, and HNB inhibits through interaction with both T1 and T2 the phosphate (Pi)-PPi transporter. Binding of CHL and HNB is freely reversible. However, the inhibition of both PPiase and Glc-6-Pase by HNB becomes irreversible following incubation of HNB-exposed microsomes with 2.5 mm sodium borohydride, indicating that inhibition involves the formation of a Schiff base. The presence of CHL effectively protects T1, but not T2, against the irreversible inhibition by HNB. In contrast, PPi and Pi are effective in protecting T2, but not T1. This is the first report describing an effective inhibitor of the system PPiase activity (T2). CHL is the most specific T1 inhibitor described to date.