Studies of interleukin 1 (IL-1) α and β have emphasized their functional similarities. IL-1α and -β are encoded by ancestrally related genes that have diverged dramatically in primary sequence; however, only modest differences in the regulation or biological activity of IL-1α and IL-1β have been documented. Here we show that mouse L cells transfected with murine pro-IL-1α cDNA expressed biologically active, 33-kilodalton pro-IL-1α, and that this pro molecule was neither processed to the 17-kilodalton mature form nor secreted. The transfected cells also expressed membrane-associated IL-1 biological activity, indicating that the pro-IL-1α cDNA can direct expression of membrane-associated IL-1 and that cleavage of the pro molecule is not required for membrane presentation. In contrast, transfection of pro-IL-1β cDNA did not generate biologically active material in L cells. Evidence is presented that the native murine IL-1β precursor molecule is also biologically inactive in peritoneal exudate cells stimulated with lipopolysaccharide. These differences in distribution of the bioactive forms of IL-1α and IL-1β may provide selective advantages for the maintenance of two gene products with similar functions.