Calcium is required to sustain fast axonal transport in sensory neurons of frog and cat. We studied the Ca2+ dependence of fast axonal transport in the motoneurons of the lower spinal cord from frog. The accumulation of acetylcholinesterase at a crush on the ventral roots was used to follow axonal transport. Two types of experiments were performed: modification of the medium bathing the ventral roots, alone, and modification of the medium bathing the spinal cord and ventral roots. Incubation (17--18 h) of the ventral roots in Ca2+-free medium markedly inhibited acetylcholinesterase transport, a finding that demonstrates a Ca2+ requirement for fast axonal transport in motoneurons; when 4 mM MgCl2 was added to the Ca2+-free medium, transport was also greatly reduced. During incubation of the ventral roots in normal medium supplemented with 0.18 mM CoCl2 transport proceeded normally; but when the Co2+ concentration was raised to 1.8 mM, transport was diminished as drastically as in the Ca2+-free medium. Incubation of the spinal cord and ventral roots in medium containing 0.18 mM CoCl2 did not reduce the accumulation of acetylcholinesterase at the crush. Similarly, accumulation of acetylcholinesterase at a crush on the dorsal root was not significantly reduced by exposure of the dorsal root ganglion and root to 0.18 mM Co2+. Exposure of sensory cell bodies to 0.18 mM Co2+ thus produces differential effects on transport of acetylcholinesterase and on transport of newly synthesized radiolabeled protein.