To identify carriers of Leptospira spp. in Argentina, wild animals were trapped in Buenos Aires Province during three nights, capturing 12 Didelphis albiventris (white-eared opossum), six Chaetophractus villosus (big hairy armadillo), five Lycalopex griseus (South American gray fox), and two Conepatus chinga (Molina’s hog-nosed skunk). All were tested by microscopic agglutination test, and five (two gray foxes, two armadillos, and one skunk) were positive for Leptospira interrogans serovars Canicola and Icterohaemorrhagiae, L. borgpetersenii serovar Castellonis, and L. kirschneri serovar Grippotyphosa, at titers of 1:50 and 1:100. Kidney tissue from all animals was cultured, and one isolate of L. interrogans from a gray fox was obtained. Hamsters inoculated with the isolate died after 6 days with no macroscopic lesions at necropsy. However, histologic examination revealed glomerulonephritis, interstitial nephritis, and pneumonia. The Leptospira strain from the South American gray fox was analyzed serologically and its pathogenicity was established. Genotyping through multiple-locus variable-number tandem repeat analysis showed that the strain was a new genotype related to the L. interrogans serogroup Icterohaemorrhagiae.