An inactive form of rabbit testis trypsin-like enzyme appears to be a zymogen. Partial purification of the zymogen is described in detail and includes extraction. Sephadex G-75 gel filtration and SE-Sephadex ion exchange column chromatography at acid pH. The zymogen was separated from a trypsin inhibitor by the Sephadex G-75 chromotographic step, but it still remained inactive unless activated with bovine pancreatic trypsin or "autoactivated" at pH 8. No enzymatic activity could be detected at pH 8 with BAEE as substrate when the zymogen was assayed immediately after pre-incubation at pH 3.