This report concerns an evaluation of three in vitro teratogenesis tests: the Dugesia regeneration assay, the Hydra reaggregation assay and the Xenopus embryo assay. Our approach involves the establishment and/or refinement of test protocols, definition of endpoints, and evaluation of test performance by comparison with available results of in vivo mammalian studies. Chemicals used for preliminary evaluation studies were the known mammalian teratogens, vinblastine sulfate (VIN) and hydroxyurea (HU), a coeffective teratogen, cadmium chloride (Cd), and an National Toxicology Program priority chemical, 9-aminoacridine hydrochloride (9AA). The Dugesia assay takes advantage of the ability of beheaded flatworms to regenerate and can be completed in 7-14 days. Concentrations of VIN of 3.2 mg 1(-1) inhibited auricle formation and further regeneration observed in 3-6 days. Similarly, eyespot and auricle formation was blocked by HU (180 mg 1(-1) ). The duration of regeneration, measured as the time elapsed between decapitation and eye-spot formation (control = 5 days), was extended by 1-4 days during exposure to 9AA. Sublethal Cd had little effect on regeneration. The Hydra assay is an evaluation of the ability of dissociated cells to regenerate complete organisms when randomly reassociated. A teratogenic test substance is detected by observing the ratio of the minimal effective concentrations of a substance between intact organisms and regenerates. Compounds with a ratio greater than 2.0 are potential teratogens. The ratios determined for 9AA, VIN, HU and Cd, respectively, were 10.0, 4.0, 2.7 and 1.2. These results indicate that the first three compounds tested positive for teratogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)