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To examine the in vivo function of IgD we generated mice deficient for IgD by gene targeting. The IgDmice show a reduced B-cell compartment with 30-50% less B cells in the spleen and lymph nodes but show a normal pre-B-cell compartment. The surface-IgD-B cells express two to three times more surface IgM than B cells ofcontrol animals. Serum concentrations of the immunoglobuln isotpes of IgDmice are almost normal, indicating that surface-IgD expression is not necessary for class switching of B cells. Immunization experiments showed that IgD-mice could respond well to thymus-dependent and -independent antigens. After immunization normal germinal centers developed in the IgD-mice. cells (7, 8). In vivo the functions attributed to the engagement of sIgD vary from the acquisition of resistance to tolerance induction (10-12) and the initiation ofthe B-cell response (13) to a role in B-cell memory (6,14,15). Some ofthese functions were studied by using chronic anti-IgD treatment from birth (16,17), by in vitro sorting of sIgD+ and sIgD-B cells (10,18,19), by enzymatic removal of sIgD in vitro (11), or by transgenic mouse models (20). Discrepancies of results were found in these studies that could be generated by the nature of the indirect experimental systems used. Roes and Rajewsky (21) PCR and Southern Blots. ES cell clones were picked and prepared for a nested primer PCR analysis, as described (35). Genomic DNA for Southern blotting was prepared from ES cells or from the tail ends of mice.Flow Cytometric Analysis (FCM). FCM of single-cell suspensions of lymphatic organs was done on a FACScan flow cytometer (Becton Dickinson). The antibodies used for staining were either (i) biotinylated and counterstained with streptavidin-phycoerythrin 11-26C (anti-IgD, from J. Kearney, University of Alabama, Birmingham) ( Fig. 2 a and b), M41 (anti-IgM) (27) (Fig. 2 c and d), B7/6 (anti-IgM) (27) (Fig. 2 g and h) or (ii) conjugated with fluorescein isothiocyanate M41 (anti-IgM) (Fig. 2 a and

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