We developed a giant colony test system with rapidly growing mycobacteria by stab-culture with a loopful inoculum of cells into Middlebrook 7H10 agar medium containing soluble extracts of the fruits of Gardenia jasminoides(7H10-crocin agar medium) and assessed the significance of the giant colony test with 28 strains of 10 clusters of rapidly growing mycobacteria classified by the simple biological 5-test characters. Of the 10 clusters of mycobacteria tested, some of strains which belonged to cluster No. 1a, 5a and 11a did grow as gravis types, whereas most of other clusters gave mitis or intermedius types in their colonial sizes at 12 days culture. By this test, pathogenic strains of M. fortuitum-chelonei complex which belonged to cluster No. 5a, b, 7a and 8a, b could be divided into gravis, intermedius and mitis colony types and the gravis ones were characterized by bluish-white "mushroom-shaped" colonies with central complexes in the texture, whereas the intermedius gave grayish-white "flower-shaped" colonies with radiated folds, but without any central complexes. The mitis colonies were characterized by grayish-white smooth or smooth mucoid colonies and were common among the clusters in their shapes. The colony of M. chelonei was bluish-white mitis type and was characterized by its hilly rhizoid colony. The gravis colony of cluster No. 1a identified as M. phlei was characterized by yellow "round straw- mat-shaped" or "chrysanthemum-shaped" colony with whole complexes in the texture, and the gravis colonies of the cluster No. 11a gave grayish-white "flower-shaped" colonies with central stamens, radiating trough and fine cup-shaped strands in the texture. The four colony types of pathogenic species of M. fortuitum-chelonei complex on 7H10-crocin agar medium were distinctive from those of other clusters of rapidly growing mycobacteria and these results indicated that the giant colony test, in conjunction with the simple 5-test characters, would be of value in the differentiation of M. fortuitum complex from other clusters of rapidly growing mycobacteria.