Whole proteins of the second-generation merozoite of Eimeria tenella were analyzed by two-dimensional gel electrophoresis (2-DE) and western blot using the chicken sera infected artificially with E. tenella. Approximately 640 spots were detected on proteome map of the second-generation merozoite stained by Coomassie brilliant blue G-250 and 85 spots were recognized on western blot map as antigens. Forty four spots of the antigens were identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) and MALDI-TOF-TOF-MS. Twenty six proteins of E. tenella and three homologous proteins to other apicomplexan parasites or protozoan were identified using 'Mascot' server. These proteins included lactate dehydrogenase, enolase, 14-3-3 protein, microneme proteins, tubulin beta chain, SERPIN1 protein precursor, large subunit ribosomal protein L23 and surface antigens of E. tenella, heat shock protein (HSP70) of Eimeria acervulina, protein phosphatase type 1 of Toxoplasma gondii and hypothetical protein GSPATT00020155001 of Paramecium tetraurelia. The rest proteins were searched against the E. tenella protein sequence database using 'Mascot in-house' (version 2.1) search engine in automated mode and 11 unknown proteins were identified. After the amino acid sequence of the unknown proteins were searched using BLAST against non-redundant sequence databases (NCBI), surface antigen 12 of E. tenella and six homologous proteins to other apicomplexa parasites were found. They were membrane skeleton protein IMC2A, mitochondrial F1-ATP synthase beta subunit precursor, 3-oxoacyl-acyl-carrier-protein synthase and catalase of T. gondii, Vps26 of Plasmodium falciparum 3D7, and hypothetical protein TRIADDRAFT_60424 of Trichoplax adhaerens. No homologues of 8 spots of the 44 analyzed proteins were found. These proteins enriched the data of immunogenic proteins of the second-generation merozoite of E. tenella.