MRC OX-22 monoclonal antibody was derived by a fusion of spleen cells from a BALB/c mouse immunized with phytohemagglutinin-activated rat lymph node cells with NS 1 myeloma cells. Rat lymph node cells were cultured in 10 gg/ml phytohemagglutinin type V from Phaseotus vulgaris (Sigma Chemical Co., Detroit, MI) in RPMI 1640 medium plus 5% DA rat serum, 2.5 x 10-5 M merca~ toethanol and 2 mM glutamine for 3 d. BALB/c mice were immunized twice with 10 blast cells per mouse intraperitoneally at an interval of 10 d, and 6 wk later were immunized again with blast cells intravenously. 3 d later the spleen cells were fused with the NS 1/1. Ag4. 1 cell line and hybrids selected according to the procedures of Galfre and Milstein (9). Cultures with hybrid cells were screened for antibody with the indirect binding assay on blast cells, and clones were obtained from positive cultures by dilution cloning with rat thymocytes as feeders. The positive cultures were recloned at intervals and after growing in mice as ascites. MRC OX-22 was found to be of lgG~ class by formation of precipitin in agar using rabbit antimouse IgGl antiserum, kindly provided by M. Parkhouse and rabbit anti-mouse IgGz~ and IgG2b serum from Miles Laboratories Ltd. Other mouse monoclonai anti-rat cell antibodies used were: W3/13 and W3/25 (4); MRC OX-7 (10); MRC OX-8 (2); MRC OX-12 (11), and MRC OX-19 (6). Rabbit anti-mouse (RAM) F (ab') 2 and its fluorescein isothiocyanate (FITC) conjugate (RAM-FITC) were prepared as described previously (4). Rat anti-rat 1 a allotype was as described in (11) and rabbit anti-rat F (ab') 2 as in (12). Antibodies were labeled with'25I by the chloramine T method to a specific activity of~ 25 uCi/ug (10).