•Five Cellulophaga algicola alginate lyases were cloned and expressed.•HPAEC-PAD/MS and HPSEC-RI were used to analyze alginate hydrolysis.•CaAly1 endolytically cleaves both PolyG and PolyM to a significant extent.•CaAly2 and CaAly3 are endolytic enzymes and mainly active on G-rich alginates.•CaAly4 and CaAly5 exolytically hydrolyze guluronic acid oligosaccharides. Alginate lyases can be used for alginate oligosaccharide production and for structural characterization or modification of alginates. For these applications it is important to obtain detailed information on mode of action and substrate specificities of alginate lyases. In this study, five alginate lyase genes were cloned from Cellulophaga algicola DSM 14237 genomic DNA, heterologously expressed, and characterized by using HPSEC-RI and HPAEC-PAD/MS. It was demonstrated that these analytical approaches can provide detailed information on preferred substrates, extent of hydrolysis, and the liberated products. The recombinant enzymes cleaved alginates endolytically (CaAly1, CaAly2, CaAly3) or exolytically (CaAly4, CaAly5). The three endolytic alginate lyases predominantly hydrolyzed guluronic acid-rich alginates, only CaAly1 also showed activity on mannuronic acid-rich alginates. The oligosaccharide profiles further demonstrated that the endolytic enzymes have rather narrow but slightly different substrate specificities and that the two exolytic alginate lyases mainly cleaved unsaturated guluronic acid oligosaccharides to monomers.