Cellular fatty acid analysis by GLC is widely used in the species identification of mycobacteria. Combining mycolic acid cleavage products with shorter cellular fatty acids increases the informative value of the analysis. A key has been created to aid in the identification of all currently known slowly growing environmental species. In this scheme, the species are classified into six categories, each characterized by a combination of fatty markers shared by those species. Within each category, individual species may be distinguished by the presence or absence of specific marker substances, such as methyl-branched fatty acids or secondary alcohols. This study also describes earlier unpublished GLC profiles of 14 rare, slowly growing, environmental mycobacteria, Mycobacterium asiaticum, Mycobacterium botniense, Mycobacterium branderi, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium doricum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium hiberniae, Mycobacterium kubicae, Mycobacterium lentiflavum, Mycobacterium scrofulaceum, Mycobacterium triplex and Mycobacterium tusciae. Though no single identification technique alone, even sequencing of an entire single gene such as 16S rRNA, can identify all mycobacterial species accurately, GLC has proven to be both reliable and reproducible in the identification of slowly growing mycobacteria. In cases of earlier unknown species, it generates useful information that allows their further classification and may lead to the description of novel species.