To characterize the genes expressed by Eimeria tenella oocysts, the sequence of 499 expressed sequence tags (ESTs) was obtained from complementary DNA (cDNAs) enriched for transcripts expressed by unsporulated or sporulated oocysts. Of these, 225 clones were isolated from cDNA of sporulated oocysts and 274 from unsporulated oocysts. A total of 163 unique sequences were found, and the majority of these (64%) represent novel genes with no significant homology to the proteins in GenBank. Approximately half of the unique transcripts generated from sporulated oocysts are also expressed by sporozoites and merozoites, whereas the expression of most (79%) of the transcripts from unsporulated oocysts has not yet been detected at other stages of development. The expression of 4 transcripts obtained from the subtracted cDNAs was confirmed by quantitative reverse transcriptase–polymerase chain reaction. The results confirmed that these transcripts are in fact differentially expressed between sporulated and unsporulated oocysts.