AbstractFertilizedRana pipienseggs were treated with 0.5% solution of trypan blue in spring water. The stage at which the dye was added and the duration of exposure were varied.The first signs of aberrant development appeared at late stage 13 at which time the neural folds were abnormal and elongation of the body axis was inhibited. Subsequent development resulted in microcephalic larvae with foreshortened trunks and kinked or malproportioned tails. Such embryos never exhibited spontaneous muscular contractions although they may survive for 10 to 12 days and come to resemble stage 21 larvae.Microscopic analysis revealed that (1) the notochord was either completely absent or present only in the anterior half of experimental embryos, (2) adjacent to the notochordal abnormalities the lateral mesoderm formed an unsegmented mass whose left and right sides fused beneath the neural tube, and (3) malformations of the forebrain included absence of the diencephalon, optic cups, and cranial ganglia.Trypan blue was never found within the cells of the embryos.The critical period of response to trypan blue extended from stage 10 through stage 14. Animals growing in dye solution since fertilization but removed to fresh spring water prior to stage 10 differentiated normally.