A second cuticlin gene, cut-2, of the nematode Caenorhabditis elegans, has been isolated and its genomic and cDNA sequences determined. The gene codes for a component of cuticlin, the insoluble residue of nematode cuticles. Conceptual translation of cut-2 reveals a 231-amino acid secreted protein which, like CUT-1, begins with a putative signal peptide of 16 residues. The central part of the protein consists of 13 repetitions of a short hydrophobic motif, which is often degenerated with substitutions and deletions. Parts of this motif are present also in CUT-1 (Caenorhabditis elegans) as well as in several protein components of the larval cuticle and of the eggshell layers of various insects (Locusta migratoria, Ceratitis capitata and Drosophila species). These sequence similarities are related to the similar functions of these proteins: they are all components of extracellular insoluble protective layers. Immunolocalisation and transcription analysis suggest that CUT-2 contributes to the cuticles of all larval stages and that it is not stage-specific. Analysis by reverse transcriptase-PCR suggests that it is not stage-specific. Analysis by reverse transcriptase-PCR suggests that transcription is not continuous throughout larval development but occurs in peaks which precede the moults. Dityrosine has been detected in the cuticle of nematodes and of insects; formation of dityrosine bridges may be one of the cross-linking mechanisms contributing to the insolubility of cuticlins. Recombinant, soluble CUT-2 is shown to be an excellent substrate for an in vitro cross-linking reaction, catalysed by horseradish peroxidase in the presence of H2O2, which results in the formation of insoluble, high-molecular weight CUT-2 and of dityrosine.