KEY WORDS: abalone larval metamorphosis, d d d d -aminovaleric acid, crustose coralline algae, Hydrolithon samoense , settlement-inducing substance.Abalone larvae swim for several days before they metamorphose into a miniature adult organism. Crustose coralline algae (CCA) induce substratumspecific metamorphosis of abalone larvae. The metamorphosis is triggered by chemical substances, such as g -aminobutyric acid (GABA) 1 and thyroid hormones, 2 but these substances have not been found as free compounds in CCA. Morse and Morse studied settlement-inducing substances from CCA and suggested that settlement was controlled by precursors or metabolites of phycoerythrin structurally related to GABA. 3 However, the active substances in the algae remained unspecified. 4 Seki et al . reported that abalone larval metamorphosis was induced by dibromomethane produced from algal surfaces. 5 The chemical structure of CH 2 Br 2 is quite different from that of GABA. The present study on inducing substances in CCA focused on low-molecular weight compounds, and resulted in the isolation of d -aminovaleric acid (5-AVA) as an inducer.Larvae of two species of abalone Haliotis discus discus and Haliotis discus hannai were supplied by Shizuoka Prefectural Sea Farming Center, Shizuoka Prefectural Thermal Effluent Utilization Research Center, and Yamagata Prefectural Sea Farming Center. Three or 4 days after fertilization at 18-20 ∞ C, the veliger larvae were transported to the University of Shizuoka. Assays were performed by slight modifications of the procedure described previously. 2 Swimming larvae, which were competent to metamorphose, were placed in 24-well tissue culture dishes at densities of 20-40/well. Assays were performed in the dark at 19 ± 1 ∞ C in 2.0 mL sterilized natural seawater containing the tested samples and 150 m g/mL each of potassium penicillin G and streptomycin sulfate (Meiji Seika Kaisha Ltd, Tokyo, Japan). Two controls were included, one containing only seawater (negative) and the other 1 m M baclofen (positive), a potent GABA B receptor agonist. 6 The percentage of metamorphosis in each well was determined at 96 h with the use of a microscope at 40 ¥ magnification. The assays were conducted in quadruplicate, and the results were shown as the mean value of four wells.Stones covered with the CCA Hydrolithon samoense were collected at Osezaki (Shizuoka Prefecture, Japan). Algae-covered stones (estimated weight of wet algae, 300 g) were directly extracted with distilled water at 4 ∞ C for 2 days. After filtering, the filtrate was lyophilized to give powdered extract (11 g). The extract was separated by a series of chromatographic methods (Fig. 1) to give an active fraction (fr-1-2-3, 20 mg). The conditions for chromatography were as follows: (i) Diaion HP-20, solvent: H 2 O AE 50% aqueous methanol AE methanol; (ii) activated charcoal, solvent: H 2 O AE 50% aqueous ethanol AE 50% aqueous ethanol containing 1% acetic acid; (iii) Amberlite CG-50 (H + form; Rohm and Haas Co., Philadelphia, PA, USA), solvent H 2 O AE ...