We have identified an open reading frame (ORF), CP530R, within the EcoRI C' fragment of the African swine fever virus (ASFV) genome that encodes a polyprotein of 62 kDa (pp62). Antisera raised against different regions of ORF CP530R recognized a polypeptide of 62 kDa in ASFV-infected cells during the late phase of virus replication, after the onset of viral DNA synthesis. Pulse-chase experiments showed that polyprotein pp62 is posttranslationally processed to give rise to two proteins of 35 kDa (p35) and 15 kDa (p15). This proteolytic processing was found to take place at the consensus sequence Gly-Gly-X through an ordered cascade of proteolytic cleavages like that which also occurs with ASFV polyprotein pp220 (C. Simón-Mateo, G. Andrés, and E. Viñuela, EMBO J. 12:2977-2987, 1993). Immunofluorescence studies showed that polyprotein pp62 is localized in the viral factories. In addition, immunoprecipitation analysis of purified virus particles showed that mature products p35 and p15 are major structural proteins. According to these results, polyprotein processing represents an essential strategy for the maturation of ASFV structural proteins.