Two species of casein kinase from lactating bovine mammary gland have been identified; a Ca 2 + - and CM-independent casein kinase and a Ca 2 + - and CM-dependent casein kinase. The Ca 2 + - and CM-independent casein kinase phosphorylates previously dephosphorylated α s 1 -, β- or κ-casein while the Ca 2 + - and CM-dependent casein kinase prefers previously dephosphorylated β- or κ-casein as substrates. Two activities are indicated by their substrate specificity, sensitivity to Ca 2 + and CM, pH maxima, and differential solubilization by anionic detergents. The presence of a regulated casein kinase in the lactating mammary gland suggests that casein phosphorylation may be a regulator of micelle formation or secretion.