The U(L)33 gene of herpes simplex virus 1 (HSV-1) encodes a protein (pU(L)33) that is essential for the cleavage and packaging of concatameric herpesvirus DNA into preformed capsids. Previous data have suggested that the U(L)33 protein interacts with the cleavage and packaging proteins encoded by U(L)15 and U(L)28 that are known to associate with capsids. Examination of purified A capsids that lack DNA and are derived from aborted packaging events, B capsids that lack DNA, and C capsids that contain DNA revealed an association of the U(L)33 protein with all three capsid types. More U(L)33 protein was detected in A capsids than was present in B capsids. Capsid association was susceptible to guanidine-HCl treatment and independent of the presence of U(L)15 or U(L)28. Capsid association of pU(L)33 was also independent of U(L)6, which is believed to encode the portal into which DNA is inserted. These data suggest that pU(L)33 may act as part of the capsid-associated molecular machinery that translocates cleaved genomic DNA into the capsid interior.