Fluorescence microscopy performed on living cells is a valuable technique for elucidating patterns of cell growth in vitro over artificial biomaterials such as vascular grafts, and for in vivo studies such as identification and treatment of atherosclerotic plaques. Two fluorescent dyes of particular value for vital fluorescence studies are Rhodamine-123 and 3,3'-dioctadecylindocarbocyanine-labeled low density lipoprotein (DiI-LDL). We examined the toxicity of these two dyes and of dimethylsulfoxide (DMSO), a solvent used in Rhodamine-123 studies, on the growth of MRC5 human fetal fibroblasts in monolayer culture. Two parameters of cell growth were quantitated: Cell number (a measure of proliferation), and cell area (a measure of cell spreading), based on microscopic images obtained at the start and end of a 48-h growth period after brief exposure (0.5 h) to test solutions. We found that the recommended solvent for solubilization of Rhodamine-123, DMSO, caused cessation of cell proliferation and actual reduction in the area covered by adherent fibroblasts at concentrations of as low as 0.1% (vol:vol). Rhodamine-123 made up from an aqueous stock solution modestly retarded proliferation and spreading, and there was no significant effect of DiI-LDL on these parameters over prolonged periods of exposure (up to 24 h) in culture. These results demonstrate that the most toxic substance for growing fibroblasts was the solvent DMSO. We conclude that both the solvent vehicle and fluorescent dye should be carefully examined for potential toxicity before such dyes are used for vital fluorescence studies of living cells.