Numerous rodents were trapped for serologic and virologic studies following the identification of a hantavirus pulmonary syndrome (HPS) case in Dade County, Florida. Cotton rats (Sigmodon hispidus) were the most frequently captured rodent and displayed the highest seroprevalence to a variety of hantavirus antigens. Hantavirus genome RNA was detected in all the seropositive cotton rats tested, using a reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay. A virus was isolated from tissues of two seropositive cotton rats by cultivation of lung and spleen homogenates on Vero E6 cells. Nucleotide sequence information obtained by direct RT‐PCR and the serologic relationships of this virus with the other hantaviruses indicate that this virus, Black Creek Canal virus, represents a new hantavirus distinct from the previously known serotypes. © 1995 Wiley‐Liss, Inc.