OBJECTIVE A biological indicator (BI), based on fluorescent detection of a spore-associated enzyme, has been developed. We evaluated the new BI against five conventional self-contained BIs in a 121 degrees C gravity-displacement sterilizer and, packaged in towel packs and disposable steam test packs, in a 132 degrees C vacuum-assisted sterilizer. METHODS In the 121 degrees C gravity-displacement sterilizer, rapid readout BIs and conventional BIs were tested together in an otherwise empty chamber. Exposure times were 5, 10, and 15 minutes. In the 132 degrees C vacuum-assisted steam sterilizer, BIs were contained in towel packs or disposable steam test packs. Surgical packs were added to represent a full load. Exposure times were 0, 1, and 2 minutes. RESULTS In gravity-displacement 5-minute cycles, the rapid readout BIs yielded 100% positive results within 30 minutes. The conventional BIs ranged from 72% to 100% positive in 48 hours. At 10 minutes, the rapid readout BIs were 83% positive after 3 hours, whereas the conventional BIs ranged from 0% to 37% positive after 48 hours. All indicators were negative after 15-minute exposures. In vacuum-assisted cycles, all indicators were negative after 3 minutes. After 1-minute exposures, the rapid readout BIs were 40% to 60% positive, whereas the conventional BIs were 15% to 20% positive. CONCLUSIONS The rapid readout BIs are a more sensitive indicator of marginal steam sterilization cycles than conventional self-contained BIs. They yield results within 3 hours, as compared to 24 to 48 hours for the conventional BIs.