Lymphokine gene expression was examined in a panel of 116 short-term murine T-lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. About 30% of clonable T cells, including both CD4+ CD8- and CD4- CD8+ cells, could be expanded for assay at an average of 22 days after cloning. By RNA blot-hybridization analysis, most clones (85-96%) expressed detectable granulocyte-macrophage colony-stimulating factor, interleukin 3, and gamma interferon mRNAs, and 11% expressed interleukin 4 mRNA. Although no differences were noted between CD4+ and CD8+ clones in the combinations of lymphokines produced, CD4+ clones on average transcribed and secreted higher levels. When the frequencies of coexpression of any pair of lymphokine mRNAs were determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. For example, among 13 interleukin 4-positive clones, 11 also transcribed gamma interferon, giving the frequency of double-positive clones expected for random association (9.6% versus 10.8%). Therefore, expression of the four lymphokine genes segregated independently among the clones and did not allow the division of T cells into subsets with distinct patterns of lymphokine synthesis.