The mycolic acids of several strains of Mycobacterium gordonae were examined by chromatographic and spectroscopic techniques. Both HPLC and TLC revealed two patterns of mycolates among the M. gordonae strains studied. As determined by TLC, one pattern was composed of alpha-, methoxy- and keto-mycolates; the other was composed of these mycolates plus an additional component, which was identified as dicarboxy-mycolates. The dicarboxy-mycolates were only found in those M. gordonae strains that displayed a so-called HPLC-double-cluster pattern. Detailed structural analyses of the dicarboxy-mycolates indicated that these compounds contained predominantly 61-65 carbon atoms (C(63) was the major component) and a trans-1,2-disubstituted cyclopropane ring. Thus, the dicarboxy-mycolate content of strains of M. gordonae determines their HPLC pattern. In spite of the differences in their HPLC patterns, and although they belonged to different PCR-restriction length polymorphism clusters, all of the M. gordonae strains examined in this study were closely related on the basis of the structural features of their alpha-, keto- and methoxy-mycolates; the predominant alpha-mycolates contained two cis-1,2-disubstituted cyclopropane rings, the major keto-mycolates contained a trans-1,2-disubstituted cyclopropane ring and the methoxy-mycolates contained one cis- or one trans-1,2-disubstituted cyclopropane ring. It is noteworthy that the strains containing dicarboxy-mycolates also displayed significant amounts of alpha-mycolates that contained one cis-1,2-disubstituted cyclopropane ring and one cis double bond. The results obtained in this study demonstrate heterogeneity among M. gordonae strains.