Several methylated analogs of fluorene were evaluated as mutagens in Salmonella typhimurium TA98 and TA100 in the presence and absence of microsomal activation. Among the methylated derivatives of fluorene assayed were 9-methylfluorene, 2-fluoro- and 2,7-difluoro-9-methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene, 2,3,9-trimethylfluorene and 2,7,9-trimethylfluorene. Mutagenic activity was observed for several of these fluorene derivatives in the presence of rat liver homogenate. The data support a previous observation that a single methyl substituent in the 9-position of fluorene is associated with mutagenic activity within this series of compounds. Substitution with fluorine at both the 2- and 7-positions of 9-methylfluorene was not associated with a loss of mutagenic activity as evidenced by the similar mutagenic activity of 2,7-difluoro-9-methylfluorene and 9-methylfluorene. However, 2,7,9-trimethylfluorene was not mutagenic under these assay conditions. 9-Methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene and 2,3,9-trimethylfluorene were active as mutagens in the presence of rat liver homogenate, but were inactive as tumor initiators when assayed on mouse skin.