The free 2-hydroxyestrone content of female rat brains was measured by two independent methods, including a direct radioimmunoassay and enzymatic conversion to stable O-methylated derivatives followed by a specific radioimmunoassay for the latter. The sensitivities of the two procedures were 10 pg and 5 pg respectively and the recoveries were greater than 85%. Neither assay method was able to detect any measurable endogenous 2-hydroxyestrone in the female rat brain at any stage of the ovulatory cycle. It is suggested that a high turnover rate of 2-hydroxyestrogens in the rat brain precludes the accumulation of detectable quantities of these metabolites in central tissues.