Rodent striatal cells were immortalized using the A58 temperature-sensitive allele of the SV40 large T antigen. Seventy-eight clones and 10 mixed cultures were characterized at the nonpermissive and permissive temperatures. Based on morphology and expression of proteins, cells were classified into three primary types, with types b and c expressing some neuronal characteristics. Type a cells have an epithelial-like morphology with coarse cytoplasmic extensions and occasional fine processes. These cells express vimentin, do not grow well under serum-free conditions and, when confluent, form a uniform monolayer. Type b cells have a polygonal shape and usually extend multiple thin processes. These cells possess large nuclei with multiple nucleoli and do not express vimentin. Type c cells have a fibroblast-like appearance, are unipolar or multipolar, and their soma is smaller than that of type b cells. Type c cells do not express vimentin, and when confluent form a uniform monolayer. Some type b and c clones express N-CAM and MAP-2. Several type b and c cell lines were found to consistently express glutamic acid decarboxylase (GAD) immunoreactivity under several tissue culture conditions. Selected cell lines were transplanted into the intact adult rat brain in several locations. Cells survived well for 15 wk and did not form tumors. The proteins expressed in vivo were similar to those expressed in vitro.