The cotransporter of Na+, K+, 2Cl–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K+ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1-mediated transport occurs as secondary active transport driven by Na+/K+-ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na+, K+, and Cl– × Cl–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na+/K+-ATPase activity is stimulated by elevated K+ or the β-adrenergic agonist isoproterenol. Extracellular K+ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 μM in well-differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β1-adrenergic antagonist betaxolol, but not by ICI118551, a β2-adrenergic antagonist. A large part of the β1-adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na+/K+-ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post-excitatory undershoot. © 2014 Wiley Periodicals, Inc.