Macroalgae of the order Laminariales (kelps) are important marine coastal primary producers with prime significance for ecosystem function. Important factors influencing their distribution include light and temperature but the molecular basis of kelp responses to these factors is poorly understood. We therefore constructed a comprehensive cDNA library from RNA sampled under various light and temperature regimes, as a basis for future studies about the mechanisms and pathways involved in acclimation to light and temperature stress in Saccharina latissima. A total of 400 503 ESTs was assembled into 28 803 contigs. We were able to assign putative functions or orthology relationships to more than 10 000 contigs by BLASTx, Interpro protein-motif annotation, or Gene Ontology (GO). The most frequent Interpro protein domains found in the cDNA library were the protein kinase-like domain, serine/threonine-protein kinase-like domain, and NAD(P)-binding and thiore-doxin-like fold domain. Enzyme code (EC) annotation yielded attributions for 480 contigs, providing a total of 625 ECs, which could be mapped to 85 biochemical pathways. Comparative genomics of S. latissima and Ectocarpus siliculosus indicated that our cDNA library gave a genome coverage of approximately 70%, assuming similar gene numbers in both species. GO term occurrence in S. latissima and E. siliculosus showed a similar distribution pattern among the root ontologies biological process, molecular function and cellular component. Comparative protein domain annotation of S. latissima und E. siliculosus showed that, probably due to the chosen stress conditions, the domains 'thioredoxin fold', 'thioredoxin-like fold', 'heat shock protein 70', and 'bromoperoxidase/chloroperoxidase C-terminal' are over-represented in the cDNA library. © 2012 British Phycological Society.