Background: Surprisingly, many inner-city residents have antibodies to Leptospira interrogans. The manner in which these persons acquire this organism in the absence of recognized occupational, recreational, or epidemic risk factors is not known. Objective: To study the epidemiology of patients with leptospirosis who acquired L. interrogans in inner-city Baltimore, Maryland. Design: Epidemiologic investigation. Setting: Inner-city university hospital. Patients: Three inner-city residents who developed leptospirosis. Measurements: Trapping rats in alleys where the patients may have acquired L. interrogans; polymerase chain reaction (PCR) analysis of patient serum and cerebrospinal fluid specimens and rat tissues to determine the presence of leptospiral DNA; and serologic testing of serum from patients and rats by microagglutination assay to confirm L. interrogans infection. Results: Three patients developed leptospirosis after probable percutaneous exposure to rat (Rattus norvegicus) urine in Baltimore alleys. A PCR assay detected L. interrogans DNA in samples of body fluid obtained from the first two patients at presentation (one in cerebrospinal fluid, the other in serum). Results of PCR done on serum drawn from the third patient after antibiotic therapy began were negative. A microagglutination test showed that all patients had high levels of antibodies to the L. interrogans serogroup Icterohaemorrhagiae. In 19 of 21 rats that were trapped in the alleys where the patients had sustained lacerations before illness developed, kidney or brain tissues were positive by PCR for the presence of L. interrogans. Conclusions: A population was discovered to be at risk for acquiring L. interrogans: urban residents who are sporadically exposed to rat urine in the inner city. Inner-city rats often carry L. interrogans. Polymerase chain reaction can quickly establish the diagnosis of leptospirosis and is useful for epidemiologic study. An endemic substrate for the transmission of the organism is present in inner-city Baltimore. Leptospirosis may become increasingly recognized in deteriorating inner cities in which rat populations are expanding.