The present study investigated the site of action of 5‐hydroxytryptamine (5‐HT) and pharmacologically characterized the receptors involved in regulating blood glucose levels in the crayfish, Procambarus clarkii. Injection of 5‐HT into intact animals increased glucose levels in a dose‐dependent manner. In contrast, 5‐HT failed to elicit a hyperglycemic response in eyestalk‐ablated animals. Effects of several 5‐HT receptor agonists and antagonists were examined. 5‐CT, oxymetazoline (both 5‐HT1 receptor agonists) and α‐methyl‐5‐HT (a 5‐HT2 receptor agonist), but not 1‐phenylbiguanide, m‐CPBG (both 5‐HT3 receptor agonists), or RS 67333 (a 5‐HT4 receptor agonist), induced hyperglycemic responses in a dose‐dependent manner. In addition, 8‐OH‐DPAT (a 5‐HT1A receptor agonist), L‐694,247 (a 5‐HT1B/1D receptor agonist), and DOI (a 5‐HT2A receptor agonist) were effective in significantly increasing the glucose levels, whereas both BW 723C86 (a 5‐HT2B receptor agonist) and m‐CPP (a 5‐HT2C receptor agonist) were ineffective. Finally, ketanserin (a 5‐HT2A receptor antagonist), but not p‐MPPF (a 5‐HT1A receptor antagonist), GR 55562 (a 5‐HT1B/1D receptor antagonist), SB 206553 (a 5‐HT2B/2C receptor antagonist), or tropisetron (a 5‐HT3 receptor antagonist), was able to block 5‐HT‐induced hyperglycemia. The combined results support the hypothesis that 5‐HT exerts its hyperglycemic effect by enhancing the release of hyperglycemic factor(s) from the eyestalks, and suggest that 5 HT‐induced hyperglycemia is mediated by 5‐HT1‐ and 5‐HT2‐like receptors. J. Exp. Zool. 286:596–605, 2000. © 2000 Wiley‐Liss, Inc.