ABSTRACT In this study a number of herpes simplex virus type 1 (HSV-1) proteins were screened, using a yeast-two-hybrid assay, for interaction with the tegument protein pUL48 (VP16). This approach identified interactions between pUL48 and the capsid proteins pUL19 (VP5), pUL38 (VP19C), and pUL35 (VP26). In addition, the previously identified interaction of pUL48 with the major tegument protein pUL36 (VP1/2) was confirmed. All of these interactions, except that of pUL35 and pUL48, could be confirmed using an in vitro pulldown assay. A subsequent pulldown assay with intact in vitro -assembled capsids, consisting of pUL19, pUL38, and pUL18 (VP23) with or without pUL35, showed no binding of pUL48, suggesting that the capsid/pUL48 interactions initially identified were more then likely not biologically relevant. This was confirmed by using a series of HSV-1 mutants lacking the gene encoding either pUL35, pUL36, or pUL37. For each HSV-1 mutant, analysis of purified deenveloped C capsids indicated that only in the absence of pUL36 was there a complete loss of capsid-bound pUL48, as well as pUL37. Collectively, this study shows for the first time that pUL36 is a major factor for addition of both pUL48 and pUL37, likely through a direct interaction of both with nonoverlapping sites in pUL36, to unenveloped C capsids during assembly of HSV-1.