The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequencedNucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the Accession number AF162691., allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35 582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Kmapp values describing interactions with dUMP (28·8 μMvs. 3·9 μM) and (6RS, αS)-N5,10-methylenetetrahydrofolate (383 μMvs. 54·7 μM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.