The RNA genome of tobacco etch virus (TEV) is expressed as a polyprotein which is co- and post-translationally processed by viral encoded proteinases. The TEV 49,000 dalton (49-kDa) proteinase cleaves the polyprotein at five positions each defined by the seven amino acid consensus sequence, (formula; see text) One of the cleavage sites, the 58-kDa nuclear inclusion/30-kDa capsid protein junction was altered by site-directed mutagenesis and the effects of these alterations on cleavage were determined. Polyprotein precursors were synthesized by translation of T7 polymerase-derived transcripts and processed in a cell-free system using TEV nuclear inclusion bodies as a source of 49-kDa proteolytic activity. A wild-type cleavage site and 61 substrates containing site-directed amino acid replacements at the nonconserved P7, P5, P4, P2, and P'2 positions were examined. Amino acid replacements flanking the putative TEV cleavage sequence at the P7 and P'2 positions had minimal effects on cleavage. Amino acid substitutions at positions P5, P4, and P2 resulted in substrates which were processed by the 49-kDa TEV proteinase, albeit generally at reduced rates. No substitution at any of these five positions resulted in total elimination of cleavage. A model is presented which proposes different roles for conserved and variable positions in the TEV heptapeptide cleavage sequence.