HeLa cells treated with 10 J/m2 of ultraviolet (UV) radiation were examined for inhibition of DNA excision by inhibitors of DNA polymerase-alpha and -beta. DNA repair synthesis and excision were inhibited by aphidicolin, a specific inhibitor of DNA polymerase-alpha. Decreased release of radioactive nucleotides from UV-damaged DNA by the inhibitor indicates that the action of DNA polymerase-alpha activity is closely associated with DNA excision. Dideoxythymidine also inhibited DNA repair synthesis but failed to block the excision, suggesting that polymerization by DNA polymerase-beta might precede removal of damaged DNA.