Three Nocardia asteroides and five Nocardia otitidiscaviarum isolates recovered from three patients with long-term nocardiosis were compared by random amplification of polymorphic DNA fingerprinting, antibiotic susceptibility testing, and enzymatic characterization. Results obtained with primer DKU49 (P. Palittapongarnpim, S. Chomyc, A. Fanning, and D. Kunimoto, J. Infect. Dis. 167:975-978, 1993) provide evidence that patient A was infected by two N. asteroides strains during a single episode of nocardiosis and that patients B and C remained infected by the same strain, respectively. Resistance to minocycline that was present in the first isolate recovered from patient B reverted to intermediate resistance in the second isolate and reverted to susceptibility in the third isolate. Resistance to penicillin G and beta-lactams was acquired by the second isolate obtained from patient C.