The secondary structure elements at the 5' nontranslated region (NTR) of the picornaviral RNAs can be divided functionally into two domains, one of which directs cap-independent translation, whereas the other is essential for viral RNA replication. For the latter, the formation of an RNA replication complex that involves particularly viral proteinase-containing polypeptides and cellular proteins has been shown (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598; Xiang W et al., 1995, RNA 1:892-904). To initiate studies on the formation of the hepatitis A virus (HAV) RNA replication complex, binding of the HAV proteinase 3Cpro and 3CD to secondary structure elements at the 5' and 3' NTR of the HAV RNA was investigated. Using mobility shift assay, UV crosslinking/ label transfer, and northwestern analysis, we show that both the HAV 3Cpro and the proteolytically inactive mutant bind to in vitro synthesized transcripts, suggesting that the RNA-binding site of the enzyme is separated spatially from its catalytic center. Weak interactions with HAV 3Cpro were found for individual secondary structure elements comprising less than 100 nt. RNA-binding specificity was unambiguous for transcripts comprising at least two stem-loops along with the polypyrimidine tract. Furthermore, competition experiments suggest that the 5' terminus of the HAV genome contains multiple binding sites for HAV 3Cpro. In contrast to poliovirus, binding capacity of HAV 3CD to RNA of the 5' NTR was not improved as compared to 3C. The data imply that, during the viral life cycle, HAV 3Cpro might serve replicative function(s) in addition to proteolysis of the viral polyprotein.