Abstract Peripheral blood lymphocytes from the Florida alligator were separated into at least two subpopulations based upon surface markers and mitogen responsiveness. Fractionation on glasswool columns yielded two subpopulations, a nonadherent one responsive in vitro to PHA and Con A but not to LPS, and an adherent one responsive to LPS (and to PHA). Depletion of cells bearing surface immunoglobulin determinants (by complement-mediated cytotoxicity and by anti-immunoglobulin affinity columns) ablated the responsiveness to LPS (and PWM) but not to PHA. LPS, but not PHA, induced a subpopulation of alligator lymphocytes to differentiate into immunoglobulin producing cells. These data, and other related studies, indicate the Florida alligator likely has T and B cells akin to those of mammals and birds.