Biological monitors (BI) are considered to be the best monitor of the sterilization process yet false positives may result in recalls and quality assurance difficulties. To assess the frequency, type and reasons for questionable results, we undertook a 4-year in-use study of two commonly used BI types—spore strips (Spordi®) and a self-contained crushable ampule (Attest®)—for both steam and ethylene oxide (EO). After laboratory verification of time/kill ratios for a portion of each involved lot, 2 BI of each type were placed in test pack within a randomly selected load run at standard time and temperature. All resulting positive BI were subcultured. Steam cycle positives were uncommon (32/1,1710 positive Spordi, 1.9%; 20/1,710 positive Attest, 1.2%) and could be related to chamber temperature or steam quality. All of the 4 BI per load were positive in only three loads; physical monitors indicated gross malfunction. Five positive Spordi were due to either contaminants or a malfunctioning incubator. EO-related positives were more common (53/1,109 positive Spordi, 4.8%; 25/1,109 positive Attest, 2.3%). One-half of the Spordi tests became positive after 48 hours of incubation. Organisms other than B. subtilis were recovered from 49.1% of the positive tests (26/53). The Attest was remarkable for its lack of contamination; 1/25 was positive for Pseudomonas stutzeri only. More positives were observed during the winter months when relative humidity was below 20%. This finding was more commonly observed with the EO Attest. In summary, we found no significant difference in the performance of either BI. Subculturing of positives and maintenance of proper humidity during storage should be emphasized.