Batten disease (juvenile neuronal ceroid lipofuscinosis) is a recessive neurodegenerative disorder of childhood. The gene, CLN3, was recently identified and found to encode a novel 438 amino acid protein of unknown function. In order to gain insight into the function of the Batten disease protein (CLN3p), we investigated its subcellular localization. Protein constructs incorporating CLN3p fused to the green fluorescence protein or an eight amino acid peptide tag were transiently expressed in fibroblasts, HeLa and COS-7 cells. A juxtanuclear, asymmetric localization pattern was observed that correlated with the Golgi apparatus in all three cell types. However, a proportion of transiently transfected cells exhibited a punctate vesicular distribution throughout the cytoplasm in addition to or without the Golgi localization. In order to account for localization patterns arising from intracellular protein transport disruption due to exaggerated overexpression in transiently transfected cells, we isolated a stably transfected cell line expressing only one copy of the CLN3-GFP DNA construct. Fluorescence and biochemical analyses using this cell line demonstrated that CLN3p is an integral membrane protein that localizes primarily in the Golgi apparatus. The functional implications of this finding are discussed.