To determine if the emergence of hantavirus pulmonary syndrome in the United States was a consequence of recent amino-acid altering mutations and/or genetic reassortment with pathogenic hantaviruses, we examined lung and spleen tissues from seropositive deer mice trapped in August 1983 in Mono County, California, for hantaviral RNA by reverse transcriptase-directed polymerase chain reaction. Alignment and comparison of 1485 nucleotides of the S and M genomic segments enzymatically amplified from these tissues indicated that these deer mice harbored a hantavirus which was genetically similar, differing by less than 2% at the deduced amino-acid level, to the hantavirus implicated in cases of hantavirus pulmonary syndrome occurring in the Four-Corners region of New Mexico, Arizona, Utah, and Colorado in 1993. The peromyscine rodent-borne hantaviruses were, in turn, genetically distinct from other well-characterized hantaviruses, diverging by approximately 30% from Prospect Hill and Puumala viruses at the nucleotide and amino acid levels. Phylogenetic analysis using the maximum parsimony, neighbor-joining, and unweighted pair-group methods indicated that the Peromyscus-derived hantavirus shared a common ancestry with arvicolid rodent-borne hantaviruses. Overall congruency of the phylogenetic trees based on the S and M genomic sequences supported the evolutionary position of the peromyscine rodent-borne hantaviruses. Our data also establish the existence of a hantavirus enzootic in deer mice long before the recognition of hantavirus pulmonary syndrome in the United States.