By using a beta-casein-derived specific peptide substrate for mammary gland Golgi-enriched-fraction casein kinase, phosphorylating activity has been detected in the Golgi apparatus of rat liver, spleen and to a lesser extent, kidney and brain, while the other post-nuclear cytoplasmic fractions are totally devoid of such a casein kinase activity. In contrast ubiquitous protein kinases CK1 and CK2 (casein kinases 1 and 2), tested with their specific peptide substrates, display different subcellular distribution and are almost undetectable in the Golgi fraction. The absence of CK2 in the Golgi fraction has been also confirmed using specific antibodies. The relatedness between the liver Golgi apparatus casein kinase (G-CK) and the bona fide mammary gland Golgi-enriched-fraction casein kinase (GEF-CK) is supported by a variety of observations, notably: (a) identical peptide substrate specificity, consistent with an S-X-E-X consensus sequence; (b) preference for Mn2+, and, to a lesser extent, Co2+, over Mg2+, as activating cation; (c) superimposable elution profiles from DEAE-Sepharose, heparin-Sepharose, and Superdex 200, this latter consistent with a molecular mass around 500 kDa; (d) insensitivity to staurosporine and heparin (a potent inhibitor of CK2) and inability to use GTP as phosphate donor (by contrast to CK2). These data provide the evidence for the existence of a third class of ubiquitous casein kinases here termed G-CK, distinct from CK1 and CK2, specifically located to the Golgi apparatus and related to the bona fide casein kinase(s) responsible for the phosphorylation of casein secreted from lactating mammary gland. The possible involvement of G-CK in the phosphorylation of secretory pathways proteins at S-X-E motifs is discussed.