Nematode parasites are of major importance in human health and agriculture, and free-living species deliver essential ecosystem services. The genomics revolution has resulted in the production of many datasets of expressed sequence tags (ESTs) from a phylogenetically wide range of nematode species, but these are not easily compared. NEMBASE4 presents a single portal onto extensively functionally annotated, EST-derived transcriptomes from over sixty species of nematodes, including plant and animal parasites and free-living taxa. Using the PartiGene suite of tools, we have assembled the ESTs publicly available for each species into a high-quality set of putative transcripts. These transcripts have been translated to produce a protein sequence resource, and each annotated with functional information derived from comparison to well-studied nematode species such as Caenorhabditis elegans and also other non-nematode resources. By cross-comparing the sequences within NEMBASE4, we have also generated a protein family assignment for each translation. The data are presented in an openly-accessible, interactive database. To demonstrate the utility of NEMBASE4, we have used the database to examine the uniqueness of the transcriptomes of major clades of parasitic nematodes, identifying lineage-restricted genes that may underpin particular parasitic phenotypes, possible viral pathogens of nematodes, and nematode-unique protein families that may be developed as drug targets.