The purpose of the experiments reported here is to provide evidence that motoneurons (MTNs) isolated from chick embryo spinal cords go through an active process of cell death when deprived of trophic support in vitro. In order to analyze and characterize this process, MTNs were isolated with a metrizamide gradient technique and cultured in the presence of saturating concentrations of soluble muscle extract. When muscle extract was washed off from the cultures, MTNs entered a process of cell death that could be blocked with inhibitors of mRNA and protein synthesis. Two other additional criteria were used to define this process as an active one. First, ultrastructural analysis of MTNs dying as a consequence of muscle extract deprivation showed that some, but not all, of the MTNs displayed clear signs of apoptotic cell death. Those included cytoplasm condensation, fragmentation of chromatin, and preservation of cytoplasmic organelles. Second, internucleosomal degradation of DNA was detected in MTNs deprived of muscle extract. When DNA was analyzed by Southern hybridization techniques using digoxigenin-labeled genomic probes, a clear ladder pattern could be identified on muscle extract-deprived MTNs. The degradation of DNA upon trophic deprivation could be prevented by cycloheximide (CHX). In an attempt to characterize further the process of active cell death in MTNs, we found a time point of commitment to cell death of approximately 10 hr by using three different approaches: muscle extract deprivation plus readdition of muscle extract, muscle extract deprivation plus addition of CHX, and muscle extract deprivation plus addition of actinomycin D. Moreover, we show that MTNs deprived of trophic support from muscle extract but maintained alive with CHX could not be rescued from cell death by reading muscle extract if CHX was washed off the cultures within the first 15 hr of muscle extract deprivation. However, muscle extract alone was able to rescue MTNs that had been kept alive with CHX for periods of time longer than 24 hr after muscle extract deprivation. From these results we postulate that the activation of the cell death program after trophic deprivation is transient.