One of the vitally important functions of glutathione (GSH) is to adequately protect cells against toxic chemicals, reactive oxygen metabolites and free radical species. The amino acid, cysteine, is the key rate-limiting substrate for the biosynthesis of GSH, and the maintenance of adequate intracellular GSH levels is dependent upon the extracellular availability and transport of cysteine into cells. In the present study, primary cultures of astrocytes and neurons were employed to characterize cysteine transport systems. Both astrocytes and neurons used Na⁺-dependent systems as the major route for cysteine uptake (80–90% of total), while Na⁺-independent uptake represented a minor component of total transport (10–20% of total). Among the Na⁺-dependent systems, X_AG⁻ was the major contributor (approx. 80–90%) for cysteine uptake in both neurons and astrocytes, with a minor contribution from the ASC transport system (Na⁺-dependent neutral amino acid transport system for alanine, serine, and cysteine). In the Na⁺-independent transport systems (10–20% of total cysteine transport), multifunctional ectoenzyme/amino acid transporter γ-glutamyltranspeptidase (GGT), and the neutral amino acid l-system contributed approximately equally towards cysteine uptake, in both neurons and astrocytes. The present studies demonstrate that astrocytes and neurons accumulate cysteine by both Na⁺-dependent and Na⁺-independent uptake systems, with major uptake occurring through the X_AG⁻ system and minor uptake via the ASC, GGT and l-systems.