Diagnosis of systemic nocardiosis has been hampered by difficulty in the rapid isolation and identification of the organism and by the frequent need for invasive biopsies. We show that a 55,000-Mr protein previously found in Nocardia asteroides is also present in N. brasiliensis and N. caviae culture filtrates. We also used the antigen in a dot blot assay to identify patients infected with N. brasiliensis. Sera from patients with mycetoma secondary to N. brasiliensis (n = 6) or Actinomadura madurae (n = 2) and 10 hospitalized controls were tested. Six (100%) of the sera from the patients with nocardial mycetoma had positive dot blots to purified antigen, whereas all 10 of the control sera were negative. Serum from one of two patients infected with A. madurae also reacted with the antigen. Thus, the 55-kilodalton protein appears to be produced by three medically important species of Nocardia and is useful when used in an immunoblot assay for identifying patients infected with these organisms.