cDNA cassettes of FMDV have been constructed which encode the capsid precursor (P1-2A) alone or with the proteases L and 3C which are required for processing of this precursor to the products 1 AB, 1 C, and 1 D. These cassettes have been analyzed using in vitro transcription and translation reactions and within cells using recombinant vaccinia viruses. Processing of the precursors occurred more efficiently in cells than in cell-free systems but similar properties were observed. It was not possible to isolate recombinant vaccinia viruses containing FMDV cassettes which included the intact coding sequence for the L protein. Deletion of part of the L sequence, which abolished its proteolytic activity, also abolished this incompatibility with vaccinia virus. The vaccinia recombinant, vTF7-3, which expresses the bacteriophage T7 RNA polymerase was used in transient expression studies using plasmids containing a T7 promoter upstream of the FMDV cassettes. Under these conditions it was possible to coexpress L, P1-2A, and 3C in the vaccinia-infected cells; each of the proteolytic activities was observed and correctly processed 1AB, 1C, and 1D were produced.