Y-1 adrenal tumor cells were grown on plastic, or plastic treated with poly(2-hydroxyethyl methacrylate) (polyHEMA) to produce concentration-dependent rounding (10(-5)-3 X 10(-4) M) of the cells or on plastic treated with poly-D-lysine (polylysine) to produce flat cells, in order to determine whether or not cell shape is correlated with steroid synthesis. The degree of rounding of cells was measured by determining mean cell height and longest cell diameter. Three measurements of steroid production were made: production of 20 alpha-dihydroprogesterone, transport of cholesterol to the inner mitochondrial membrane, and production of pregnenolone by isolated mitochondria. Cells grown on poly(HEMA) showed increase in mean cell height, decrease in longest diameter (. rounding), and increase in all three measurements of steroidogenesis. In the case of synthesis of 20 alpha-dihydroprogesterone, the response was dependent on the concentration of poly(HEMA), being greater with higher concentrations (up to 10(-4) M), of this agent. Moreover the degree of rounding (cell height) was correlated with production of 20 alpha-dihydroprogesterone at three concentrations of poly(HEMA) (r = . ACTH at a supramaximal concentration produced increases in all of these responses to the poly(HEMA) surface. Polylysine produced flatter cells (lower mean height and greater longest diameter) than plastic and also inhibited all three steroidogenic responses to ACTH. (Bu)2cAMP exerted the same effects as ACTH. Growing cells on poly(HEMA) or polylysine did not affect production of cyclic AMP by the cells. Addition of poly(HEMA) or polylysine to the medium in which the cells were incubated, at the same concentrations as those used for influencing cell shape, was without effect on steroid synthesis or the response to ACTH. Cells grown on poly(HEMA) show decreased incorporation of [3H] thymidine into DNA. It is concluded that cell shape influences the delivery of cholesterol to inner mitochondrial membrane and in this way, increases the production of steroids by Y-1 cells and that the effects of poly(HEMA) on cell shape, cholesterol transport, and synthesis of DNA may involve microfilaments.