Five 3-alkylpyridine alkaloids, amphimedosides A-E (1-5), have been isolated from a marine sponge Amphimedon sp. The structures of 1-5 have been determined by analysis of NMR and FABMS data and chiral GC analyses of the acid hyrolyzates. In particular, the site of glycosylation in 1 was confirmed by the 1H-15N HMBC experiment, and the location of the double bond in 5 was assigned on the basis of tandem FABMS data. Amphimedosides are the first examples of beta-D-glucosylated 3-alkylpyridine alkaloids and exhibited cytotoxic activities comparable with those of the nonglycosylated congeners.