As a first step in understanding the function of the 68-kDa Alz-50 antigen (A68) in the pathophysiology of Alzheimer's disease (AD), we have reexamined preliminary observations in our laboratory (Wolozin and Davies, 1986) of a protein kinase activity associated with crude preparations of the protein. This study was undertaken to determine whether the kinase activity is an inherent property of the Alz-50 antigen, or is a property of an associated protein. Phosphorylation was therefore examined by incubating A68-enriched preparations with radiolabelled ATP. This resulted in the appearance of a labelled 68-kDa phosphoprotein, comigrating with the Alz-50 reactive A68 protein. The labelling of this 68-kDa protein occurred in the presence of 2% SDS, suggesting that it is more likely to represent an autophosphorylation than a transfer of phosphate mediated by another kinase. Upon further inspection, it was found that the autophosphorylated 68-kDa protein was not localized to regions of AD brain where A68 was detectable, but displayed a more ubiquitous distribution. In addition, this phosphoprotein was also observed to be present in similar preparations from normal brain, which lacked the Alz-50 antigen (Wolozin et al, 1986). These findings indicate that the auto-kinase activity at 68 kDa is not closely associated with the A68 protein, but with a comigrating contaminant in the preparation. Other experiments in this study indicate that A68 is not a substrate for in vitro phosphorylation. Following incubation of A68 preparations with radiolabelled ATP, immunoprecipitation of the antigen did not reveal any phosphate transfer to the protein. These results were unaffected by a prior incubation with alkaline phosphatase, even when the subsequent phosphorylation reactions were conducted in the presence of protein kinase activators. Incubation with alkaline phosphatase did not produce any alterations in electrophoretic mobility of A68, nor did it affect the binding of antibodies directed against phosphatase-sensitive epitopes with A68. Thus, despite the suggestion that A68 is a modified form of tau, the antigen exhibits remarkable differences from tau with regard to its sensitivity to kinases and to alkaline phosphatase.