Isolation and characterization of BALB/c lymphocytes specific for phosphorylcholine is described. The isolation protocol utilizes phosphorylcholine coupled to gelatin coated plates via the cleavable crosslinking reagent N-succinimidyl 3-(2-pyridyldiothio)propionate (SPDP). The procedure is rapid, requiring only 1-2 h and conducted entirely at 0-4 degrees C. Hapten binding cells are eluted by vigorous pipetting at 4 degrees C with medium containing 20% fetal calf serum. Approximately 70% of isolated cells rebind antigen as assessed using a PC Brucella abortus rosette assay while 50% express the TEPC15 idiotype. Approximately 85% of isolated and idiotype positive cells are B cells while the remainder are T cells. Limiting dilution analysis revealed that approximately 1/5 of PC binding cells isolated from the spleens of normal mice respond to lipopolysaccharide plus dextran sulfate by production of anti-PC antibody. Approximately 1/11 respond to PC Brucella abortus and 1/250 respond to PC sheep erythrocytes plus primed T cells by anti-PC antibody production. The results clearly demonstrate the effectiveness of this technique for isolation of highly enriched, functional, antigen specific lymphocytes.