Abstract The fused dihydrofolate reductase/thymidylate synthase gene of Toxoplasma gondii contains ten exons spanning ~8 kb of genomic DNA. We have examined the ends of DHFR-TS transcripts within this gene, and find a complex pattern including two discrete 5′ termini and multiple polyadenylation sites. No TATAA box or other classical promoter motif is evident in 1.4 kb of genomic DNA upstream of the coding region, but transcript mapping by RNase protection and primer extension reveals two prominent 5′ ends at positions −369 and −341 nt relative to the ATG initiation codon. Upstream genomic sequences include GC-rich regions and the (opposite strand) WGAGACG motif previously identified in other T. gondii promoters. Mutagenesis of recombinant reporter plasmids demonstrates that this region is essential for efficient transgene expression. Sequencing the 3′ ends from multiple independent mRNA clones demonstrates numerous polyadenylation sites, distributed over >650 nt of genomic sequence beginning ~250 nt downstream of the stop codon. Within this region, certain sites seem to be preferred: 14 different positions were found among the 32 polyadenylated transcripts examined, but ~40% of the transcripts map to two loci. The 3′ noncoding region is rich in A and T nucleotides, and contains an imperfect 50 nt direct repeat, but no obvious poly(A) addition signal was identified.