Apoptosis plays a major role in motor neuron survival during developmental cell death, after axotomy, and in motor neuron diseases. Bax is the first member of the bcl-2 family shown to promote apoptosis. In the present study, we used the bax-deficient mouse model to determine the role of bax in motor neuron survival in vitro by using dissociated spinal cord cultures. This system enables the maturation of individual motor neurons in a controlled in vitro environment. Motor neurons were identified by using the antineurofilament antibody SMI-32 and the antitranscription factor antibody Islet1. Both antibodies labeled large motor neurons in wild-type and bax-null cultures. Differentiated wild-type cultures exhibited a reduction in long-term cultures of two- and fivefold in the number of SMI-32- and Islet1-positive cells, respectively. The reduction in the number of motor neurons was attenuated in bax -/- cultures. Bax deficiency also attenuated serum withdrawal- and kainate-induced apoptosis in motor neurons. For comparison, necrotic cell death led to significant motor neuron cell death in both wild-type and bax -/- cultures. In addition, bax deficiency did not induce proliferation of motor neuron precursors in vitro. This study indicates for the first time that bax has a dominant role in the survival of long-term cultured motor neurons.