By using a subtractive hybridization strategy, we have identified a gene (TC26) that is expressed in metacyclic and tissue culture derived trypomastigotes of Trypanosoma cruzi but not log stage epimastigotes and is induced during the differentiation of metacyclic stages in vitro. In contrast, the TC26 transcript is absent from stationary phase epimastigotes of a strain that fails to undergo metacyclogenesis under the same culture conditions. Transcription of TC26 can be induced in epimastigotes by incubation with cyclic AMP and cyclic AMP analogues but it is inhibited by activators of cAMP dependent phosphodiesterases. Cyclic AMP fails to enhance tubulin gene expression in the same parasites. While present in the genome in multiple copies, the TC26 gene is expressed as a single mRNA species of approximately 5 kb. Computer analysis of the sequence of a 650-bp cDNA clone revealed no significant homologies at either the nucleotide or amino acid levels with other known proteins. Possible roles for the TC26 gene product in metacyclogenesis are discussed.