The intracellular (IC) and extracellular (EC) pools of interleukin 1 (IL-1) of human monocyte cultures were found to differ in their molecular size and charge characteristics. EC activity was found by Sephadex G-75 chromatography to consist mainly of a single peak in the 15,000-17,000 mol. wt range. In contrast, IC activity was distributed in four peaks (mol. wts of approx. 15,000, 26,000, 45,000 and greater than 70,000). Treatment of a pool of the IC 26,000, 45,000 and greater than 70,000 mol. wt species with CHAPS, a zwitterionic detergent, yielded a large amount of the 15,000 mol. wt species, thus suggesting that a portion of the larger species consists of aggregates of the 15,000 mol. wt molecule. Both IL-1 pools were found by isoelectrofocusing to be composed of three molecular species with pIs of 5.5, 6.7. However, the proportions of these species differed markedly between the EC and IC pools. The large majority of IC activity (approximately 90%) was found at pI 5.5, while 55-60% of EC activity had a pI of 6.7 and 35-40% had a pI of 5.5. The differences in their biophysical properties support the notion that the IC and EC pools of IL-1 also differ in their functions.