Objective: By analyzing and comparing results from a 2008 proficiency test (PT) event to results from a 2003 PT event, we assessed whether laboratories’ abilities to detect Escherichia coli ( E. coli ) O157:H7 had improved in the intervening 5 years. Methods: A proficiency test sample containing E. coli O157:H7 was mailed to participants enrolled in a PT program, and a survey about stool culture screening practices was distributed to laboratories that had submitted a result for the proficiency test sample. Results: In 2008, 85.3% of laboratories detected E. coli O157:H7, compared to 70% of laboratories in 2003. Also, 72.3% of laboratories now screen at least all bloody stools, compared to 49% of laboratories in 2003. Conclusions: Laboratories’ abilities to detect E. coli O157:H7 has improved substantially since 2003. However, many laboratories still fail to follow screening recommendations for Shiga toxin-producing E. coli .