Nocardia isolates that share the property of in vitro amikacin resistance are grouped together by some authors in the Nocardia transvalensis complex. Our examination of 13 isolates that are amikacin resistant has revealed the existence of three distinct species. Sequence analysis of the 16S rRNA, 65-kDa heat shock protein, and secA1 genes, coupled with DNA-DNA hybridization, indicated that "N. asteroides drug pattern IV," "N. transvalensis new taxon 1," and N. transvalensis sensu stricto should each be considered a distinct species. The phenotypic and molecular characteristics of the proposed new species Nocardia wallacei (N. asteroides drug pattern IV) and N. blacklockiae (N. transvalensis new taxon 1) are presented and compared with those of N. transvalensis sensu stricto. The relative genetic diversity of isolates best placed with the species N. blacklockiae is also discussed. Case studies demonstrating the pathogenicity of N. wallacei and N. blacklockiae are presented. The type strain of N. wallacei is ATCC 49873 (DSM 45136), and that of N. blacklockiae is ATCC 700035 (DSM 45135).