Interleukin-1 (IL-1) is synthesized as a 31 kDa precursor protein, whose multiple extracellular activities are attributed to receptor binding of a processed, carboxy-terminal 17 kDa peptide. Unlike other secreted proteins, the IL-1 precursor lacks a hydrophobic leader sequence and is not found in organelles composing the classical secretory pathway. In order to further clarify the intracellular processing of IL-1, we studied its site of synthesis in human monocytes. Secreted and integral membrane proteins are translated on membrane-bound polyribosomes, while intracellular proteins are translated on free polyribosomes. Free and membrane-bound polysomes were isolated from Lipid A-stimulated monocyte lysates and immunoblotted using antibodies specific to the N-terminal regions of the IL-1 alpha and beta precursors. Free polysome fractions showed multiple small bands consistent with nascent peptide chains; membrane-bound polysomes yielded no detectable IL-1. Polysome fractions were then analyzed by immunoelectron microscopy; nascent IL-1 alpha and beta peptide chains were readily seen emerging from cytoskeletal-associated free polyribosomes, but not membrane-bound polyribosomes. Electron microscopic in situ hybridization revealed IL-1 mRNA chains attached to cytoskeletal-associated free, but not membrane-bound polyribosomes. The intracellular distribution of the fully synthesized IL-1 beta precursor was studied in human mesangial cells (HMC), whose cytoskeletal organization is more readily evaluated than that of monocytes. Dual immunofluorescence microscopy of these cells revealed a complex intracellular distribution of the fully synthesized 31 kDa IL-1 precursors. IL-1 was asymmetrically distributed between cytosolic, microtubule, and nuclear compartments, without association with actin or intermediate filaments. This demonstration of the sites of IL-1 synthesis and patterns of intracellular distribution provide further evidence for an extracellular release mechanism which is clearly distinct from the classical secretory pathway.