In an effort to explain the difference in potencies between the two characterized crustacean pigment-dispersing hormones (alpha-PDH; beta-PDH) and to define a role for residue 3 in these octadecapeptide hormones, we have synthesized and purified seven position 3 alpha-PDH analogues ([Ala3], [Ile3], [Asn3], [Gln3], [Asp3], [Glu3], and [Lys3]alpha-PDH). When tested for melanophore pigment-dispersing activity in destalked Uca, [Glu3]alpha-PDH was found to be 325% more potent than alpha-PDH. Reduced potencies were observed for the [Asp3] (58%), [Asn3] (26%), [Gln3] (11%), and [Ala3] (8%) derivatives. Much lower potencies were displayed by the [Lys3] and [Ile3] analogues (0.73% and 0.66%, respectively). These results suggest that the position 3 side chain carboxylate anion of [Glu3]alpha-PDH stabilizes the active receptor-bound conformer through a charge-charge interaction.