A series of phenothiazine, benzo[a]phenothiazine and benz[c]acridine derivatives were compared for their ability to induce nucleosome-sized DNA fragmentation (a biochemical hallmark of apoptosis), using agarose gel electrophoresis and a fluorescence activated cell sorter. Significant DNA fragmentation-inducing activity was detected in 12H-benzo[a]phenothiazine, 5-oxo-5H-benzo[a]phenothiazine and 9-methyl-12H-benzo[a]phenothiazine, which induced the monocytic differentiation of human myelogenous leukaemic cell lines. On the other hand, an other three benzo[a]phenothiazines, six 10-[n-(phthalimido)alkyl]-2-substituted-10H-phenothiazines, six 1-(2-chloroethyl)-3-(2-substituted-10H-phenothiazin-10-yl)alkyl-1- ureas, and twelve benz[c]acridines showed little or no DNA fragmentation-inducing activity. Active benzo[a]phenothiazines induced DNA fragmentation in four human myelogenous leukaemic cell lines (HL-60, ML-1, U-937, THP-1), but not in human T-cell leukaemic MOLT-4 and erythroleukaemic K-562 cell lines, which were also resistant to other apoptosis-inducing agents. Ca2+-depletion from the culture medium did not significantly affect their DNA fragmentation-inducing activity. The differentiation and apoptosis-inducing activity of benzo[a]phenothiazines have an important role for their medicinal efficacy.