Four anti-peptide antibodies were raised, each against a synthetic peptide corresponding in sequence to a short stretch of crustacean hyperglycemic hormone (CHH) or CHH-L (CHH-like) peptides of the crayfish Procambarus clarkii; CHH and CHH-L are alternatively spliced products that share an identical sequence for the first 40 residues from the amino-terminus of the peptides. When used in Western blot analyses of tissue proteins, anti-CHH (1-10) recognized an immunoreactive protein band in both the sinus gland (SG) and thoracic ganglia (TG), whereas anti-D-CHH (1-10) recognized an immunoreactive protein band only in SG, but not in TG; anti-CHH (59 - 72) recognized an immunoreactive protein band in SG but not in TG, and conversely, anti-CHH-L (58 - 72) recognized an immunoreactive protein band in TG but not in SG. Tissue homogenates were fractionated using high performance liquid chromatography (HPLC). The collected HPLC fractions were determined for immunoreactivity by enzyme-linked immunosorbent assay and Western blotting, and the immunoreactive fractions subjected to mass determination. A pair of stereoisomers － CHH and D-Phe3 CHH, both with a mass of 8386.4, and immunoreactive to anti-CHH (1-10) and anti-D-CHH (1-10), respectively, was identified in SG; Western blot analyses showed they were immunoreactive to anti-CHH (59 – 72), but not to anti-CHH-L (58 – 72). A CHH-L, with a mass of 8343.6 and immunoreactive to anti-CHH (1-10) but not to anti-D-CHH (1-10), was identified in TG. Western blot analyses showed it was immunoreactive to anti-CHH-L (58 – 72), but not to anti-CHH (59 – 72), and sequencing analysis of the peptide fragments generated by enzyme digestion of the immunoreactive protein revealed 3 sequences, which are contained within a CHH-L encoded by previously identified transcript. Further, the anti-peptide antibodies were tested for the effects on blocking CHH-induced hyperglycemia. Results showed anti-CHH (59 - 72) and anti-CHH (1-10) individually abolished the CHH-induced hyperglycemia, whereas control treatments, pre-immune sera or anti-CHH-L (58 - 72), did not significantly affect CHH-induced hyperglycemia. In summary, these data reiterate the observations that CHH and CHH-L are preferentially expressed in different tissues; they also suggest that enzyme(s) involved in L-to-D isomerization of CHH is expressed in a tissue-specific manner. Finally, the data suggest the N- and C-terminal regions of CHH are important for its biological activity.