Though bilirubin is reported to affect a variety of cellular functions, the primary target of its toxic effect is still not known. A major problem in understanding this is the wide variation in results reported by different groups. This is probably due to the differences in stability of bilirubin solutions arising from large differences in bilirubin:albumin molar ratios used in experiments. Hence in studying the toxic effects of bilirubin in tissue culture systems, it is important to be certain that the bilirubin is maintained in solution throughout the time of the exposure to bilirubin. Spectrophotometric measurements have shown that bilirubin is stable in Dulbecco's modified Eagle medium solution at bilirubin:albumin molar ratios up to 3. Under these defined conditions, bilirubin was found to affect Na+K+ ATPase, [3H]-thymidine uptake, L-[35S]methionine incorporation into protein and mitochondrial function at bilirubin concentrations up to 125 microM and bilirubin:albumin molar ratio of 1.5. Toxic effects on all parameters measured were evident at bilirubin:albumin molar ratio of 1.5 after a minimum of 2 h of exposure. No effect was evident at a bilirubin:albumin molar ratio below 1. Although it is not possible to identify with certainty the primary target, the effect on mitochondrial function appeared earlier and was more profound than that seen with the other assessed functions.