Biochemical, genetic, and morphological criteria were used to demonstrate that human cytomegalovirus penetrates permissive fibroblasts and nonpermissive Chinese hamster ovary (CHO) cells by pH-independent fusion between the virus envelope and the host cell plasma membrane and not by low pH-induced fusion within endosomes. Viral immediate early (IE) gene expression and infectivity were unaffected by conditions which block various stages of endocytosis or agents that alter the acidic pH of the endosome. IE gene expression was also evident in a mutant CHO cell line which is defective in endosomal acidification. Morphological analysis of the entry process at the electron microscopic level revealed viral particles in various stages of virion-plasma membrane fusion. In contrast, intact enveloped virions were not observed sequestered within coated pits or vesicular structures. Collectively, the data indicate that the entry pathway by which HCMV gains access to the cytoplasm of fibroblasts and CHO cells in order to initiate infection is via pH independent, virion envelope-plasma membrane fusion.