We have previously shown that tooth development is a sensitive endpoint of dioxin toxicity. In the present study we determined the critical window of sensitivity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity for the development of rat molar teeth. Dams were exposed to 1 microg/kg once over a time range covering the development of each molar tooth. A cross-fostering study also was conducted. Pups were killed at the age of 40 days. Erupted molars were observed by stereomicroscopy. The jaws were radiographed, the presence of molars assessed from the radiographs, and the molars measured. Pups exposed both in utero and via lactation lacked third molars and the frequency of missing molars was greater the earlier the dam was exposed: 100, 88, and 50% of the offspring exposed on gestation day (GD) 11, GD13, and GD19, respectively. All third molars developed in offspring of dams exposed on postnatal day (PND) 0, PND2, or PND4. None of the exposed pups lacked the first or the second molars. In the cross-foster study, 32 and 4% of offspring exposed only in utero and only via lactation, respectively, lacked molars. TCDD accelerated the eruption of the lower incisors and retarded the eruption of the third molars. The most critical window of sensitivity for the development of the third molar was during the early morphogenesis, from tooth initiation to the early bud stage, after which the sensitivity substantially decreased. Response to TCDD was increased if the exposure was started several days before this critical window of sensitivity or if it was continued thereafter. Dental epithelium is the likely target of TCDD.