The cytotoxic activity of 4''-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), a novel acridine derivative with clinical antitumor activity, was examined in multicellular spheroids grown from Chinese hamster [lung] V79-171b cells. m-AMSA is much less effective against cells within these tumor-like structures than it is against exponential-phase V79-171b cells in monolayer cultures, the initial DO [mean lethal dose] of the survival curve for the latter being approximately 10-fold lower than for the former following a 60 min exposure to the drug. The resistance of spheroid cells to m-AMSA appears to be at least partially a result of the noncycling or slowly cycling state of the majority of these cells, although they are more sensitive than cells in plateau-phase monolayers. A further component of resistance in spheroids requires the presence of an intact spheroid structure and may be due to drug transport limitations. The use of sequential trypsinization techniques to recover cells at varying depths within spheroids demonstrates that a 60 min m-AMSA treatment preferentially kills cells nearest the spheroid surface, suggesting that tumor cells at a distance from the vasculature may limit the efficacy of m-AMSA in vivo.