High-density cultures of mouse embryo limb bud cells differentiate and synthesize an extracellular matrix containing sulfated proteoglycans. Since these in vitro events are related to those that occur during fetal development, we have investigated the use of cultured limb bud cells for the analysis of the activities of chemical teratogens. We have established the conditions for the use of the radiochemicals 35SO=4 and 3H-thymidine for the assessment of chemical effects on sulfated proteoglycan and DNA synthesis, respectively, in mouse limb bud cells. By performing double-labeling experiments in the presence of test chemicals, the preferential inhibition of either proteoglycan synthesis or DNA synthesis could be demonstrated for 19 of 22 known mouse teratogens tested. The five nonteratogens tested did not cause significant differences in the inhibition of either macromolecular class. The overall predictive accuracy of the system was approximately 89%, and the false-negative rate was approximately 14.8%. No false positives were observed. These data showed that this cell culture system differentiated between the activities of a limited set of selected mouse teratogens and nonteratogens, suggesting that cultured mouse embryo limb bud cells may constitute the basis for the development of a powerful tool for analyses of chemical teratogenic potential.