SUMMARY: The amount of coagulase formed in vitro by coagulase-positive staphylococci depends on: the age of the bacterial cells, coagulase being produced only during the latter part of the lag-phase of growth; the period of incubation of the bacterial cells under ideal conditions, the optimum period depending upon the age of the cells when incubation starts; the initial concent ration of bacteria. It also depends on the presence in the medium of a fermentable sugar and a factor found in brain-heart infusion and partially replaceable by peptone; the salt concentration in the medium; the initial pH value of the medium; the temperature of incubation: and upon substances present in rabbit serum and, to a lesser extent, horse serum, human serum and egg yolk, egg white and commercial nucleic acid not showing this effect. The activity of rabbit serum is destroyed by treatment with pepsin. Provided that the requisite conditions are met, cell-free coagulase which clots rabbit plasma within a few minutes, can be prepared in less than one hour.