To understand the physiological role of the calcium-binding proteins S100α and S100β, it is necessary to determine the distribution of these proteins and detect their intracellular targets in various tissues. The distribution of immunoreactive S100α and S100β in various rat tissues was examined by radioimmunoassay. All tissues examined contained detectable S100, but the S100β/S100α ratio in each tissue differed. Brain, adipose, and testes contained 18- to 40-fold more S100β than S100α; skin and liver contained approximately equivalent amounts and kidney, spleen, and heart contained 8- to 75-fold more S100α than S100β. Analysis of S100-binding proteins by gel overlay showed that each tissue possessed its own complement of binding proteins. The S100β-binding profile was indistinguishable from the S100α-binding profile and both of these profiles were distinct from the calmodulin-binding profile. These observations suggest that the differential distribution and quantity of the individual S100 polypeptides and their binding proteins in various tissues may be important factors in determining S100 function.