Epstein Barr virus (EBV) immortalizes human B lymphocytes, resulting in long-term lymphoblastoid cell lines. To date, such cell lines have typically been derived by suspension culture of mixed mononuclear cells, infected in vivo or in vitro with EBV. We have derived 208 lymphoblastoid lines by clonal transformation of peripheral blood lymphocytes from 3 neonates, 3 normal adults, and 1 patient with systemic lupus erythematosus (SLE), utilizing colony formation in agarose and then separate propagation of the colonies in liquid medium. A total of 180 lines have been analyzed for expression of cytoplasmic and/or secreted immunoglobulin isotypes. Such analysis reveals that 167 (92%) of the lines are monoclonal with respect to isotype expression. All clonally derived lines from neonates produce only IgM. Most clonal transformants from normal adults also produce IgM (81%), but clones producing IgG (16%) or IgA (3%) were also found. Clonal transformation of lymphocytes from the SLE patient revealed a shift in isotype expression, with increased numbers of IgG-secreting clones (48%) and IgA-secreting clones (13%). The data support the use of EBV-mediated clonal transformation as a probe for the analysis of states of isotype differentiation among circulating B cells, and as a method for deriving immortalized, monoclonal human B cell lines.