Black Creek Canal (BCC) virus is a newly identified virus associated with hantavirus pulmonary syndrome (HPS) in the southeastern United States. Nucleotide sequences were determined for the complete S and M and partial L genomic segments of a BCC virus isolate. Phylogenetic analysis indicates that each of the virus segments is unique, and there is no evidence of genetic reassortment having occurred between this and other previously characterized hantaviruses. All hantavirus-seropositive, wild-caught Sigmodon hispidus which were tested by polymerase chain reaction for the presence of BCC virus RNA were found to be positive, consistent with a chronic virus infection. This finding, together with serologic evidence, confirms S. hispidus as the primary rodent reservoir of this virus. Nucleotide sequence analysis of these PCR products indicates that BCC virus is genetically diverse and may pose a public health threat throughout much of the extensive range of this rodent species. Immunoprecipitation analysis demonstrated the presence of G1-, G2-, and N-specific antibodies in sera from BCC virus-infected patients with HPS and naturally infected S. hispidus. Comparison of the patterns of protein immunoprecipitation obtained with human acute- and convalescent-phase sera suggests that G1- and G2-specific antibodies increase relative to N-specific antibodies during the course of infection. The ratio of G1 to N protein immunoprecipitation by rodent or human acute-phase sera allowed differentiation of BCC and Sin Nombre virus infections.