Plasma membrane chloride (C1⁻) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active C1⁻ uptake mechanism) in C1⁻ handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin‐perforated patch‐clamp recordings revealed that DRGNs accumulate intracellular C1⁻ through a bumetanide‐ and Na⁺‐sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of CU during GABA_A receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting CU concentration (i.e., [C1⁻]_i decrease) was similar in both cell types and was unaffected by lowering the extracellular Na⁺ concentration. In contrast, the recovery on depletion (i.e., [C1⁻]_i increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na⁺. The experimental observations could be reproduced by a mathematical model for intracellular CU kinetics, in which DRGNs show higher NKCC1 activity and smaller C1⁻‐handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [C1⁻]_i, whereas lower NKCC1 activity in MNs allows large [C1⁻]_i variations during neuronal activity.—Chabwine, J. N., Talavera, K., Verbert, L., Eggermont, J., Vanderwinden, J.‐M., De Smedt, H., Van Den Bosch, L., Robberecht, W., Callewaert, G. Differential contribution of the Na+‐cK+‐2Cl− cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons. FASEB J. 23, 1168–1176 (2009)