DNA sequence analysis of the short unique regions in the genomes of herpes simplex virus (HSV), types 1 and 2, has previously shown that within this region there are four genes, designated US2, US4, US5 and US7, whose functions are unknown but whose predicted amino sequences exhibit hydrophobic N-termini (D.J. McGeoch et al., 1985, J. Mol. Biol. 181, 1-13; D.J. McGeoch, H.W.M. Rixon and D. McNab, unpublished data). In this paper, the possibility was investigated that these hydrophobic sequences might be signal sequences associated with membrane-bound translation of the proteins, and subsequent secretion or insertion into membranes. By using reference sets of protein sequences known to be translated either on membrane-bound or on free ribosomes, criteria were developed to distinguish between these two classes. These criteria comprised: length and net charge of the immediately N-terminal region which often precedes the hydrophobic stretch in membrane-translated proteins; length of the uncharged (hydrophobic) region; and degree of hydrophobicity of the 8-residue maximal hydrophobic region. The latter two parameters were found to be particularly effective when combined as a two dimensional plot, which clearly distinguished 96% of membrane-translated proteins from other classes. When the uncharacterized, predicted HSV protein sequences were judged by these tests, it was found that the products of genes US4, US5 and US7 were convincingly classified as membrane-translated, while the US2 product gave a less definitive result. In conclusion, the US4, US5 and US7 gene products were considered probably to be previously unrecognized, virion membrane-inserted glycoproteins.