The U_L3 open reading frame (ORF) has the coding capacity of 235 codons. The proteins reacting with the anti-U_L3 antibody form in denaturing polyacrylamide gel bands with apparent M_rs of 34,000, 35,000, 38,000, 40,000, 41,000, and 42,000 and designated 1 to 6, respectively. Studies on their origins revealed the following. (i) The U_L3 proteins forming all six bands were present in lysates of cells infected with wild-type virus and treated with tunicamycin or monensin or in cells infected with the mutant lacking the gene encoding the U_S3 protein kinase. (ii) The proteins contained in the slower-migrating bands were absent from cells infected with the mutant lacking the U_L13 protein kinase. Bands 1 and 3, however were phosphorylated in cells infected with this mutant. (iii) Band 2 protein was absent from cells transfected with a plasmid carrying a substitution of the predicted first methionine codon of the U_L3 ORF and superinfected with the U_L3 mutant. (iv) Band 1 and 3 proteins were absent from lysates of cells transfected with a plasmid carrying a substitution of the second (M12) methionine codon of the U_L3 ORF and superinfected with the U_L3 mutant. (v) Cells superinfected with mutants lacking both methionine codons did not accumulate any of the proteins contained in the six bands. (vi) In vitro transcription-translation studies indicated that the translation of band 1 protein was initiated from the second (M12) methionine codon and that band 3 protein represented a U_L13-independent, posttranslationally processed form of these proteins. The results indicate that the U_L3 protein of herpes simplex virus 1 is translated predominantly from the second in-frame methionine codon and is subject to at least two posttranslational modifications.