The effect of adriamycin (1 mg/liter) on the development of the golden hamster 3-day-old second maxillary molars (M2) was investigated in vitro. Exposure of the molars to 1 mg/liter adriamycin during the first 2 hours of culture produced smaller teeth 3-7 days later, as determined by measurements of dry weights and by histological observations. Higher doses caused severe necrosis. The more differentiated pulp fibroblasts showed osteodentin formation 3 days after treatment with adriamycin (1 mg/liter), while the more immature ones underwent necrosis. The phenotypic changes brought on by the drug were permanent, and osteodentin continued to be formed throughout the course of this study. In addition the cervical loop region was inhibited from growing, while the production of the matrices of enamel and dentin appeared to be increased at 3 and 5 days after treatment. Electron microscopy of the forming osteodentin matrix revealed a random arrangement of banded collagen fibers during the early stage of osteodentin formation. As more matrix was formed, the collagen became quite compact and appeared quite similar to dentin. Finally, matrix vesicles were found among the collagenous matrix that was not yet mineralized. With the exception of the increased production of enamel and dentin, these in vitro results confirmed those earlier in vivo studies on the effect of adriamycin on rat incisor tooth.