Nonimmunized guinea pigs possess rare lymphocytes which bind sufficient 2,4-dinitrophenyl-guinea pig albumin-(125)I (DNP-GPA) to their surface to be detected by short-term radioautography. The cells occur in the lymph nodes, spleen, peripheral blood, and bone marrow with a frequency of approximately 40/100,000 lymphocytes, but are absent from the thymus. The receptors of these cells are largely specific for the haptenic group (epsilon-DNP-L-lysine) as shown by inhibition of DNP-GPA-(125)I binding with epsilon-DNP-L-lysine and with DNP bovine serum albumin (DNP-BSA). Furthermore, these cells specifically adsorb to agarose beads to which either DNP-GPA, DNP-BSA, or DNP-keyhole limpet hemocyanin (KLH) has been covalently linked. This hapten specific depletion of DNP-GPA-(125)I antigen-binding cells (ABC) correlates with a similar diminution in the capacity of adsorbed populations to transfer primary responsiveness to DNP-KLH to irradiated syngeneic recipients. Fluoresceinated anti-immunoglobulin binds to the surface of some guinea pig lymphocytes, and all DNP-GPA-(125)I ABC, as shown by a double-label technique. The great majority of DNP-GPA ABC and human gamma-globulin ABC possess surface Ig molecules of the gamma(2) heavy chain class. Preincubation of cell suspensions with anti-gamma(2) antibody markedly diminishes the number of DNP-GPA-(125)I ABC which are detected, strongly suggesting that the receptors of these cells are immunoglobulin molecules, most of which possess gamma(2) heavy chains. The specificity characteristics of DNP-GPA-(125)I ABC are strikingly different from those of cells mediating a cellular immune response to DNP-GPA, indicating major differences in the specificity and nature of the receptors of these cell types.