With the recognition of the importance and the prevalence of herpes simplex virus infections and the availability of effective chemotherapy, there has come a demand for rapid, sensitive, and accurate diagnosis. Virus isolation in cell culture remains the most widely employed diagnostic method, and the cell cultures used vary greatly from laboratory to laboratory. Therefore, a study was undertaken to compare several commonly employed human fibroblast cell strains and two nonhuman cells, primary rabbit kidney and guinea pig embryo cells, for the primary isolation of this virus. Our experience in the isolation of herpes simplex virus in the clinical laboratory with these cell systems was evaluated, and reisolations from clinical specimens were performed. We found that guinea pig embryo and rabbit kidney cell cultures were equally sensitive for the isolation of herpes simplex virus from clinical specimens and were superior to the human embryonic lung fibroblast cell strains tested in terms of both sensitivity and rapidity of appearance of cytopathic effect. The use of guinea pig embryo cells for the isolation of this virus from clinical specimens has not been previously reported.