AbstractA putative choline transporter (CHOT1) has been cloned from rat brain and is reported to express a high‐affinity, sodium‐dependent, hemicholinium‐3‐insensitive choline transporter in oocytes. A second transporter (OCCREATRA) cloned from rabbit brain is 98% homologous to CHOT1 and is reported to transport creatine. We examined the distribution of CHOT1 mRNA in rat brain by in situ hybridization, using a 48 base oligonucleotide probe. In adult rats, the hybridization signal was widespread, but with a distinct pattern. High levels of expression were detectedin the cerebellum (Purkinje and granule cell layers), choroid plexus, medial habenula, pontine nuclei, several brainstem, nuclei, and hippocampus (pyramidal cell layer). Moderate signal was detected in cortex, globus pallidus, corpus callosum, and most other white matter tracts. Very low levels were present in striatum, nucleus accumbens, hippocampus molecular layer, and cerebellar molecular layer. Emulsion autoradiography indicated cellular localization to both neurons and glia. CHOT1 mRNA was relatively abundant in some cholinergic regions, including the medial habenula, the medial septum, and several brainstem nuclei. However, the overall pattern was distinctly different from that expected for cholinergic markers and correlated well with the localization of creatine kinase. The widespread distribution and poor correlation with cholinergic markers indicates that the CHOT1 gene does not encode the classical choline transporter known to be associated with acetylcholine synthesis. It is possible that CHOT1 is associated with cholinergic neurotransmission in some brain regions. However, it appears to encode the rat creatine transporter, and its widespread and heterogeneous distribution suggests regions where creatine phosphate is an important energy source. © 1995 Willy