Mutagen damage to a cell located at some distance from the site of application of the mutagen will depend, in part, on how effectively it can penetrate through cells composing different tissues. Chinese hamster V79 spheroids were used to model mutagen "penetrability" by providing several layers of cells growing with tissue-like packing. By treating cells of intact and trypsin-dissociated spheroids at the same drug-to-cell ratio, an estimate relative sensitivity to nine mutagens was determined. The effective toxicity index (ETI) was defined as the ratio of the concentrations of mutagen required to kill 50% of cells (measured after a 1-hr treatment at 37 degrees C) in dissociated versus intact spheroids and similarly for mutation at the HGPRT locus (EMI). Values for ETI were consistent with values for EMI and varied from 0.01 for 4NQO to 2 for AF-2. Direct information on mutagen penetration was obtained for fluorescent mutagens by flow cytometry. Values for EFI (effective fluorescence index) varied from 0.07 for Hoechst 33342 to 1 for AF-2. These results can be interpreted as reflecting differences in ability of mutagens to penetrate spheroids; some direct-acting mutagens are likely to be effective only near their site of application (ie, UV, Hoechst 33342, 4NQO) while others are able to penetrate through successive cell layers with little difficulty (ie, monobromobimane, AF-2).