Molecular investigation of two Nocardia patient isolates showed unusual restriction fragment length polymorphism patterns with restriction endonuclease assays (REA) using an amplified portion of the 16S rRNA gene. Patterns typical of Nocardia nova were obtained with REA of an amplified portion of the 65-kDa heat shock protein gene. Subsequent sequence analysis of the 16S rRNA gene regions of these isolates showed the presence of ambiguous bases within an expected restriction endonuclease recognition site which were not able to be resolved on repeat testing. Cloning of amplified regions of the 16S rRNA genes and subsequent sequencing of the resulting clones from the two patient isolates showed three different 16S rRNA gene sequences which corresponded to sequences found in N. nova, a molecular variant of N. nova, and a previously undescribed sequence. Hybridization studies using a DNA probe corresponding to an 89-bp conserved region of the 16S rRNA gene confirmed the presence of at least two copies of the 16S rRNA gene in the N. nova type strain, in a patient isolate identical to the molecular variant of N. nova, and in the two other patient isolates. All isolates were found to belong to the species N. nova as determined by DNA-DNA hybridization. Because minimal variation has been found in the 16S rRNA gene sequences of different species of Nocardia, those laboratories employing molecular methods for identification of these species must be aware of the potential identification complications that may be caused by the presence of differing 16S rRNA genes in the same isolate.