The usefulness of an in vitro assay for embryotoxicity may depend on the availability of metabolic activation systems that will function in the culture system. The fetal mouse salivary gland has been investigated as an in vitro assay system. To see if the glands would grow in the presence of metabolic activators and if the glands would react to metabolites known to be embryotoxic, the glands were grown in the presence of cyclophosphamide (CP) and several activation systems. These included isolated rat hepatocytes, uninduced rat S-9, rat S-9 induced with 3-methylcholanthrene (3-MC), rat S-9 induced with Aroclor 1254, and human S-9. Twenty salivary glands were isolated from 13 day embryos (plug day = 0) and were grown in each treatment for 48 h. One control had no activation system of CP, one had an activation system but no CP, and three treatments had the activation system and 25, 75, or 150 micrograms/ml CP. The S-9 with cofactors and the appropriate amount of CP was contained in dialysis bags. The greatest suppression of salivary gland growth occurred in co-culture with hepatocytes activating CP. The S-9 induced by Aroclor 1254 was nearly as effective as the hepatocytes. The next most effective was a group with similar activity consisting of the uninduced rat S-9 and the three samples of human S-9. The 3-MC-induced S-9 was the least effective in suppressing growth of salivary glands. All the activation systems tested can be used with the salivary gland culture system.