A previously undescribed larval settlement-inducing protein was purified from adult extracts of the barnacle, Balanus amphitrite (=Amphibalanus amphitrite). Results of SDS-PAGE indicated that the relative molecular mass of the protein in reduced and denatured form is 31,600 +/- 500 kDa, and that it is distinct from the Settlement Inducing Protein Complex (SIPC) which has previously been determined as a larval settlement-inducing pheromone. The N-terminal 33-residue sequence of the intact protein showed no similarity with previously reported proteins in the EMBL/Genbank/DDBJ databases. The purified protein at a concentration of 10 microg ml(-1) induced approximately four times more larval settlement than the control (filtered natural seawater). In addition, results of the assay using both 24-well polystyrene plates and agarose gels indicated that this protein is probably released into seawater and attracts cypris larvae. These results suggest that the purified protein is a waterborne type pheromone which induces settlement of larvae of B. amphitrite.