Many teleostean fish, including rainbow trout, regulate red blood cell (RBC) pH (pH(i)) in the presence of a stress-induced acidosis such as hypoxia, hypercapnia, or exhaustive exercise. This is accomplished through activation of RBC Na+/H+ exchange (beta-NHE), ultimately minimizing impairment to oxygen transport. Presence and characterization of the RBC beta-NHE in fish is best tested in blood from cannulated, resting animals; however, several studies have used blood from stressed animals drawn from the caudal vein and stored prior to use. The effects of sampling procedures and storage on the beta-NHE response is not known and is the focus of this study. Whole blood drawn from cannulated, resting rainbow trout was compared with RBCs obtained from the caudal vein rinsed and stored at 4 degrees C for 0, 6, 24, 48, 96 or 144 h. Isoproterenol (10(-5) M), a beta-adrenergic agonist, was added to hypoxia/hypercapnia incubated RBCs in vitro. In all treatments, isoproterenol induced a large beta-NHE response, and storage duration (< or =96 h) had a minimal affect, indicating that rinsing and storing is an easy and viable means by which to obtain RBCs and investigate function. Storage for 144 h still resulted in a significant RBC beta-NHE response; however, viability of RBCs may be compromised.