Treatment with adriamycin for 8-14 h irreversibly induces K562 human erythroleukemic cells to synthesize hemoglobin. With 16-h exposure, this effect is maximal at concentrations between 180 and 400 nM, yielding 70%-90% benzidine-positive (B+) cells and 24 pg/cell hemoglobin 4 days after the beginning of adriamycin treatment. This induction is accompanied by changes in ouabain-sensitive 86Rb influx opposite to those seen with murine erythroleukemic (MEL) cells. Amiloride and several amiloride analogues strongly inhibit adriamycin induction of hemoglobin synthesis as well as cell growth in the absence of adriamycin. The inhibition of induction is enhanced with the analogues bearing a benzyl or substituted benzyl group on the 5-amino or on a terminal guanidino nitrogen atom. The effect on growth was somewhat greater with the analogue bearing a 2-chlorobenzyl moiety on a terminal guanidino nitrogen atom and with the one bearing a 2-fluorobenzyl group on the 5-amino nitrogen atom. The structural features required for growth inhibition resemble those seen with MEL cells, but the features required for inhibition of induction of hemoglobin synthesis are completely different. These data suggest that different specific binding sites are involved in these two effects of amiloride and its analogues.