Bacteriophage lambda and plasmid clones containing African swine fever virus (ASFV) DNA inserts, which together covered more than 90% of the genome of a Malawi ASFV isolate (LIL 20/1), were transfected into vaccinia virus (VV)-infected cells. Expression of ASFV-encoded proteins was assayed at late times after VV infection by immunoprecipitation of [35S]methionine-labeled proteins with hyperimmune serum from ASFV-infected pigs, separation of immunoprecipitated proteins by denaturing polyacrylamide gel electrophoresis, and detection by autoradiography. Synthesis of eight additional proteins not observed in control experiments was detected. Seven VV recombinants were constructed, each containing an ASFV DNA insert from a separate bacteriophage lambda clone ranging in size from 9 to 15 kb. BSC40 cells were infected with recombinant viruses and expression of ASFV-encoded proteins assayed at early and late times postinfection. Synthesis of additional proteins, not observed in control experiments, was detected by immunoprecipitation with ASFV antiserum both early and late postinfection with two of these recombinants. In these experiments VV promoters were not included upstream of individual ASFV genes.