The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.