Protein III is a neuron-specific phosphoprotein which consists of two polypeptides, IIIa (Mr 74 kD) and IIIb (Mr 55 kD). This phosphoprotein has previously been shown to be associated with synaptic vesicles. In the present investigation, we have examined Protein III in human brain tissue. In contrast to observations in rat brain, where only one form of Protein IIIb (Mr 55 kD) has been found, in human brain tissue three variants of Protein IIIb, designated Protein IIIb1 (Mr 55 kD), Protein IIIb2 (Mr 57 kD) and Protein IIIb3 (Mr 59 kD), were observed by both biochemical and immunochemical assays. Protein IIIa from human brain also exhibited three variants in electrophoretic mobility. Peptide maps of Proteins IIIa and IIIb revealed that the differences in electrophoretic mobility of the variants of these proteins were preserved in variants (Mr 18kD, Mr20kD, and Mr22kD) of a smaller peptide fragment. These variant forms of Protein III were studied in brains from individuals without any history of neurological or psychiatric illness, as well as from individuals who had suffered from one of several types of neuropathological conditions, such as chronic alcoholism, Alzheimer's disease, multi-infarct dementia, and Parkinson's disease. Some differences were observed in the distribution of variants among the various clinical categories.