The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus. About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.