To understand better how spontaneous motoneuron activity and intramuscular nerve branching influence motoneuron survival, we chronically treated chicken embryos in ovo with either d-tubocurarine (dTC) or muscimol during the naturally occurring cell death period, assessing their effects on activity by in ovo motility measurement and muscle nerve recordings from isolated spinal cord preparations. Because muscimol, a GABA(A) agonist, blocked both spontaneous motoneuron bursting and that elicited by descending input but did not rescue motoneurons, we conclude that spontaneous bursting activity is not required for the process of normal motoneuron cell death. dTC, which rescues motoneurons and blocks neuromuscular transmission, blocked neither spontaneous nor descending input-elicited bursting and early in the cell death period actually increased burst amplitude. These changes in motoneuron activation could alter the uptake of trophic molecules or gene transcription via altered patterns of [Ca(2+)](i), which in turn could affect motoneuron survival directly or indirectly by altering intramuscular nerve branching. A good correlation was found between nerve branching and motoneuron survival under various experimental conditions: (1) dTC, but not muscimol, greatly increased branching; (2) the removal of PSA from NCAM partially reversed the effects of dTC on both branching and survival, indicating that branching is a critical variable influencing motoneuron survival; (3) muscimol, applied with dTC, prevented the effect of dTC on survival and motoneuron bursting and, to a large extent, its effect on branching. However, the central effects of dTC also appear to be important, because muscimol, which prevented motoneuron activity in the presence of dTC, also prevented the dTC-induced rescue of motoneurons.