The role of calcium in neurulation in mammalian embryos has been studied by culturing rat embryos at 10.4 days of gestation, when the cephalic neural folds have elevated but not fused, in serum containing cytoskeletal inhibitors or calcium antagonists. The effects of these antagonists on the morphology of the cephalic neural folds have been examined by scanning electron microscopy. The different agents caused the cephalic neural folds to part to varying degrees. The neural folds were classified as intact (normal), open (folds parted up to 90 degrees with each other), flattened (folds parted from 90 degrees to 180 degrees) or collapsed (folds parted more than 180 degrees). The microtubule inhibitors colchicine and nocodazole at 10(-4) M respectively cause the cephalic neural folds of 10.4-day embryos to collapse after 60 min. At 5.2 X 10(-6)M the microfilament inhibitor cytochalasin B causes the folds to open after 60 min. Longer term culture of 9.5-day embryos for 24 h in diazepam, which is reported to inhibit myosin synthesis, causes general developmental retardation including a delay in the closure of the neural tube. Culture of 10.4-day rat embryos for 60 min in papaverine at 2.4 X 10(-4) M or gallopamil (D-600) at 5.0 X 10(-4) M, agents which reduce the entry of calcium into cells, causes opening of the elevated cephalic neural folds. In contrast TMB-8, which is purported to perturb some intracellular calcium-dependent functions, does not cause opening of the elevated cephalic neural folds, even at high concentrations. The results suggest that both microtubules and microfilaments are essential to the maintenance of the elevated cephalic neural folds in rat embryos. The results are also compatible with the idea that calcium ion flux across the membranes of the neuroepithelial cells might be important for the elevation of the neural folds, and thus for successful neurulation.