Two novel sphingosine-derived azetidine alkaloids, penaresidin A, 1a, and B, 1b, were isolated as potent actomyosin ATPase activators from the Okinawan marine sponge Penares sp. and the structures elucidated on the basis of spectral data, especially two-dimensional NMR spectra of their acetates. In our continuing studies on bioactive substances from marine organisms, we have investigated extracts of numerous marine invertebrates collected in Okinawa, and the bioassay-guided purification resulted in the isolation of multifarious compounds with intriguing structures and interesting biological activities, some of which may have useful clinical applications or be useful as chemical probes in the life sciences. Recently we have examined extracts of the sponge Penares sp. and have isolated a novel antileukaemic triterpenoid, penaesterol. Further inquiry into the bioactive constituents of this sponge led to the isolation of two novel azetidine alkaloids, which we have named penaresidin A (compound 1a) and B (compound 1b), possessing potent actomyosin ATPase-activating activity. This is the first isolation of sphingosine-derived azetidine alkaloids from marine sources. In this paper we describe the isolation and structure elucidation of compounds 1a and 1b.