Abstract: Disclosed are mammalian thromboplastin reagents and methods for preparing such reagents. The thromboplastin reagents are suitable for use in prothrombin-time (PT) assays, and offer improved sensitivities with acceptable PT-normal times. Contaminating proteins—particularly plasma clotting factors—are separated from a thromboplastin solution by a membrane permeation protocol, in which the thromboplastin solution is exposed to a semipermeable membrane and clotting factors are allowed to pass from the thromboplastin solution through the membrane, while Tissue Factor is retained in the thromboplastin solution. In a preferred method, one or more contaminating clotting factors are separated by diafiltration from a NaCl/Na3Citrate thromboplastin extract of mammalian tissue, and the purified thromboplastin extract is combined with Ca++ ions to form a thromboplastin reagent.