As a model for the effect of hormones and growth factors on three-dimensional growth of mammalian cells, we have analyzed the effect of insulin on the three-dimensional growth and morphology of Chinese hamster ovary (CHO) colonies grown on the surface of agar. Sequential photographs in dark-field illumination of growing colonies have been analyzed with computer-assisted techniques. In this analysis the entire shape of each colony in a sizeable population (up to 10(5) colonies per experiment) can be measured and distributions of parameters derived from these measurements can be studied. In fetal calf serum (FCS), insulin has a dose-related stimulatory effect on cell growth that is most pronounced when growth has slowed down. In 10% FCS, insulin has a similar but diminished effect. When CHO cells are grown conventionally on plastic substrata or in suspension, insulin has little effect on cell growth at 4% serum concentration. Computer analysis of changes in the distribution of colony morphology proved to be a sensitive, dose-dependent, and reproducible assay of a hormonal effect. As little as 5 ng of insulin per ml added to 10% FCS causes a shift in the distribution of colony morphologies. In 4% FCS, 50 ng of insulin per ml is required to produce a detectable change in the colony morphology distribution. Computer analysis of cells grown three-dimensionally on agar provides a powerful approach to studying the effects of hormones and provides observations not available when cells are grown on plastic substrata.