1. The time sequence of the development of acetylcholinesterase (AChE), acetylcholine (ACh) receptors and functional synapses on the embryonic muscle membrane in a tunicate larva (Halocynthia roretzi) was investigated in vivo.2. The fertilized tunicate egg was incubated in natural sea water at 9 degrees C. Sixty-eight hr after fertilization the free-swimming larva was hatched, which had six striated muscle fibres in the tail. The developmental stage of the embryo was indicated by the developmental hours after fertilization.3. The transmitter at the neuromuscular junction in the hatched larva is ACh. (i) Neuromuscular transmission was completely blocked by D-tubocurarine (1-5 x 10(-5)M). (ii) Eserine (5-10 x 10(-7)M) approximately doubled the time constant of the falling phase of miniature excitatory junctional currents (e.j.c.s). (iii) The reversal potential of the membrane response to iontophoretically applied ACh was -10 mV and similar to that of e.j.c.s. (iv) AChE was present on the muscle membrane surface.4. AChE activity became visible histochemically on the embryonic cell membrane in the presumptive muscle region as early as the late gastrula stage (27 hr after fertilization, 12 hr before the ACh response appeared).5. The response to iontophoretically applied ACh was present at 39 hr after fertilization but could not be evoked at 38 hr.6. Between 39 and 41 hr after fertilization, the ACh responses increased rapidly, then remained relatively unchanged until larval hatching.7. The stage of the initial appearance of the ACh response corresponded to the stage when the Ca current abruptly increased in the muscle membrane.8. The first sign of neuromuscular transmission was appearance of a giant excitatory junctional potential (e.j.p.) with uniform amplitude (about 15-20 mV) and slow time course (time constant of the falling phase of a giant e.j.c. was 23.4 +/- 6.9 msec, mean and S.D., at -60 mV and 11 degrees C).9. Within a few hours, these giant e.j.p.s disappeared and were successively replaced by medium-sized e.j.p.s and then e.j.p.s similar to those seen in hatched larvae (time constant of the falling phase of a miniature e.j.c. was 8.5 +/- 1.8 msec at -60 mV and 11 degrees C).