Recent studies have shown that mast cells (MCs) are present in rat brain, that they have a predominantly thalamic localization, and that they contain histamine (HA). However, the degree to which these cells contribute to brain HA levels has remained unclear. Our recent studies of the precise distribution of rat brain MCs permitted us to develop a method to determine both the MC numbers and HA content from the same brain. Thalamic MC numbers were highly correlated with both the amount (ng) and the concentration (ng/g) of thalamic HA in both sexes (p less than 0.005). Slopes of these regression lines, suggestive of the HA content of thalamic MCs, were 2.5 and 1.3 pg/cell in males and females, respectively, substantially less than the HA levels in peritoneal MCs. Thalamic MC numbers were not correlated with HA (ng) outside of thalamus, but were significantly (p less than 0.005) correlated with whole brain HA amounts (ng) and levels (ng/g). These results are direct biochemical evidence for a contribution by MCs to brain HA levels, and indicate that thalamic MCs contribute up to 90% of the HA in thalamus, and up to 50% of whole brain HA levels.