Hemolymph gas parameters were evaluated using a point-of-care analyzer in healthy American horseshoe crabs (Limulus polyphemus) at rest and after short- and long-term removal from water. Baseline vascular pH, partial pressure of oxygen (PO₂), partial pressure of carbon dioxide, bicarbonate, base excess, total carbon dioxide, and lactate concentrations were determined from hemolymph samples collected from 10 horseshoe crabs (group 1) submerged in water and were compared with values after removal from water for 5 min, and after recovery in water for 10 min and for longer than 60 min (range, 61–221 min). Hemolymph gas parameters were also determined in 12 horseshoe crabs (group 2) after shipment out of water for 24 hr and were compared with values obtained from group 1 animals. Baseline hemolymph gas values of the American horseshoe crab are within range for other aquatic vertebrates. After removal from water for 5 min, all group 1 crabs developed severe hypoxia, with PO₂ levels falling below the detectable limit of the analyzer. Group 2 crabs had pronounced respiratory acidosis, and their PO₂ values were significantly below baseline values of group 1 animals.