At early developmental stages in the rat spinal cord (embryonic day 13), when neuronal progenitors are still proliferating, most differentiating neurons express truncated forms of glutamic acid decarboxylase (GAD) (approximately 25 kDa) which are the products of alternative splicing of the GAD67 gene. These truncated proteins do not appear to synthesize gamma-aminobutyric acid (GABA). The amino acid is detected in cells only after alternative splicing of the GAD67 gene generates a full-length, 67 kDa enzymatically active form of GAD. Both the 67 kDa GAD and GABA colocalize and appear diffusely distributed in the cytoplasm of embryonic neurons. GABA does not appear associated with synaptic vesicles until after birth, when its intracellular distribution becomes punctate and it colocalizes with synaptophysin. At this time, it also colocalizes with an immunologically distinct 65 kDa GAD protein encoded by a second GAD gene (GAD65). Expression of different GAD-related proteins with distinct intracellular distributions during development suggests that GABA, the product of these enzymes, may have trophic or metabolic roles during spinal cord differentiation.