Root avulsion of adult spinal nerves causes the subacute cell loss of motor neurons. To explore the mechanisms of the elimination of motor neurons, we investigated the expression of two molecules—neuronal nitric oxide synthase (nNOS) as a cytotoxity marker and a 27-kD heat shock protein (HSP27) as a cytoprotection marker—in rat spinal motor neurons after ventral root avulsion, using immunofluorescent labeling technique for confocal laser microscopy. A drastic cell loss of motor neurons occurred during the first week following the avulsion, and the surviving motor neurons fell to approximately 60% of the control value at one week. Subsequent cell loss proceeded slowly, as the surviving motor neurons decreased to 35% at nine weeks. HSP27 immunohistochemistry showed that normal spinal motor neurons consisted of two types of motor neurons: HSP27-negative small motor neurons (<500 μm²) (about 30%), and HSP27-positive large motor neurons (>500 μm²) (about 70%). At one week, all of the HSP27-negative small motor neurons had died and only HSP27-positive large motor neurons survived. This event was followed by the induction of nNOS in the surviving large motor neurons, which showed a significant upregulation of HSP27. HSP27-negative small motor neurons were thus found to be more vulnerable to avulsion than HSP27-positive large motor neurons, suggesting that HSP27 may have protected the avulsed motor neurons from cell death. In addition, NO was involved in the gradual cell death of large motor neurons. The persistent upregulation of HSP27 and its colocalization with nNOS in surviving motor neurons may imply a keen competition in motor neuron survival between cytotoxic and cytoprotective systems.