The expression of the CD3zeta subunit was investigated in fresh (uncultured) tumor-infiltrating lymphocytes (TILs) isolated from either solid tumor (ST) specimens or ascites (ASC) from patients with epithelial ovarian carcinoma (EOC). Western blot analysis of CD3zeta immunoprecipitates using anti-CD3zeta rabbit serum revealed that in 6 out of 6 patients with EOC, the CD3zeta protein was absent from ST-TILs. Immunoprecipitation with anti-phosphotyrosine monoclonal antibody (anti-PY20) from ST-TILs from one patient revealed bands co-migrating with the phosphorylated CD3zeta. CD3zeta protein was found to be expressed in only 1 out of 7 ST-TILs from patients with EOC. ASC-TILs were available in 5 of these patients and immunoprecipitation/Western blotting experiments using anti-CD3zeta rabbit serum revealed that CD3zeta protein was expressed in all 5. In addition, CD3zeta protein was expressed in 3 additional ASC-TIL specimens for which ST-TILs were not available. Therefore, the CD3zeta protein was expressed in ASC-TIL isolated from 8 out of 8 patients with EOC. CD3zeta protein was also expressed on peripheral blood mononuclear cells (PBMCs) from patients with EOC and from normal donors. RT-PCR studies of fresh ST-TIL specimens, using CD3zeta-specific primers, revealed that CD3zeta transcripts were absent from 13 out of 21 patients with EOC, down-regulated in 4 patients and present at levels comparable to those found in PBMCs in 4 other patients. In contrast, CD3delta transcripts were present at comparable levels in all specimens. Treatment with recombinant interleukin-2 (rIL-2) (600 IU/ml) restored the expression of CD3zeta protein and transcripts in cultured ST-TILs, whereas fresh ST-TILs did not express CD3zeta, in contrast to fresh ASC-TILs. These results demonstrate differential expression of CD3zeta in ST-TILs versus ASC-TILs in patients with EOC. CD3zeta transcripts and protein were found to be absent from most ST-TILs from patients with EOC, whereas they were expressed in ASC-TILs and PBMCs from such patients.