The accuracy of 18S rRNA, β-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, β-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of β-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated γδTCR+, CD4+ and CD8+ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30–70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.