Constructs with alterations in the normal order and spacing of polyadenylation sites of the mouse Ig-gamma 2b heavy chain gene were transfected into J558L cell tumor lines (myelomas) and A20 B cell tumor lines (lymphomas) representative of plasma and memory cells, respectively. When the membrane-specific (mb) polyA site was moved from its 3'-location to a position upstream (5') of the secretory (sec) polyA site, the mb site was used preferentially, even though the sec site was still efficiently transcribed. The relative strength of the mb polyA site seems to preclude efficient use of downstream elements. When two sec polyA sites are put in competition with each other in the same transcript, use of the first site predominates in both cell types, implying that the relative strength and the distance between the sites are important for normal regulation. When the sec polyA site is put upstream of the mb polyA site, in the absence of a competing splicing event, the sec polyA site is used preferentially in the myeloma cell but not the lymphoma cell, implying that its use is a regulated event. We therefore conclude that the B cell-regulated strength of the sec polyA site, as well as its 5'-location, relative to the unregulated, but very strong mb polyA site, are important parameters in the regulated expression of mb and sec mRNA in this system.