Replicase gene expression by the human coronavirus 229E involves the synthesis of two large polyproteins, pp1a and pp1ab. Experimental evidence suggests that these precursor molecules are subject to extensive proteolytic processing. In this study, we show that a chymotrypsin-like enzyme, the virus-encoded 3C-like proteinase (3CL^pro), cleaves within a common region of pp1a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kDa are processed from pp1a/pp1ab by proteolysis of the peptide bonds Q3546/S3547, Q3629/S3630, Q3824/N3825, and Q3933/A3934. Relative rate constants for the 3CL^pro-mediated cleavages Q2965/A2966, Q3267/S3268, Q3824/N3825, and Q3933/A3934 were derived by competition experiments using synthetic peptides and recombinant 3CL^pro. The results indicate that coronavirus cleavage sites differ significantly with regard to their susceptibilities to proteolysis by 3CL^pro. Finally, immunoprecipitation with specific rabbit antisera was used to detect the pp1a/pp1ab processing end products in virus-infected cells, and immunofluorescence data that suggest an association of these polypeptides with intracellular membranes were obtained.