Functional and phenotypic properties of porcine peripheral blood CD4/CD8 double-positive (DP) lymphocytes were examined. In cross-sectional and longitudinal studies involving a total of 103 pigs, this lymphocyte population was found to increase gradually in proportion with age, comprising < 2% of the total peripheral blood lymphocyte pool in 1-week-old swine and reaching 30-55% by 3 years of age. CD4/CD8 DP lymphocytes were able to proliferate in response to stimulation with recall viral antigen. Furthermore, these cells mostly expressed high levels of the surface antigen recognized by monoclonal antibody (mAb) 4B4 (4B4hi), which is specific for the human beta 1 integrin. The CD4+4B4hi lymphocytes from pseudorabies virus-immune swine, proliferated in response to stimulation with the homologous virus, while CD4+4B4lo lymphocytes did not. Stimulation of CD4 single-positive (SP) cells with recall viral antigen, but not with mitogen, resulted in the generation of lymphoblasts which were predominantly of CD4/CD8 DP phenotype, suggesting a role for recall antigen in the generation of this lymphocyte subset. More than half of the CD4+ lymphocytes from palatine tonsils of 6-month-old swine were CD4/CD8 DP, while in the lymph nodes CD4/CD8 DP cells accounted for only one-third or less of CD4+ cells. In contrast, CD4/CD8 DP lymphocytes were absent from the palatine tonsils of 3-day-old swine, which only contained CD4 SP cells. Together, these results indicate that porcine CD4/CD8 DP lymphocytes, exhibit properties of mature antigen-experienced cells, and are inducible by stimulation with recall antigen. These data are consistent with the hypothesis that this population in swine includes memory/effector T cells.