The growth of multicellular tumor spheroids, MTSs, from squamous carcinoma line MDA 886Ln was inhibited by beta-all-trans retinoic acid (RA). Inhibition occurred within 3 to 5 days of treatment, and MTS size then remained static for up to 2 weeks. Although their growth stopped, 10-day-treated MTSs incorporated [3H]thymidine into trichloroacetic acid-precipitable material, and the [3H]thymidine labeling index, determined by autoradiography, was equivalent between control and RA-treated MTSs. Bivariate flow cytometric analysis of bromodeoxyuridine-labeled MTSs showed equivalent S phase progression of labeled cells over an 8-hour chase. MTS growth stasis was not related to RA-induced cell cycle effects. Monitoring of MTSs for cell sloughing showed no significant cell shedding that could account for stasis. Quantitation of cell number and DNA content per MTS showed an RA-induced decrease. This was confirmed by histological analysis, which demonstrated the temporal appearance of acellular areas. MTS growth statis is thus related to an RA-induced cell loss in this MTS model for squamous carcinomas.