Biological specimens from pilots fatally injured in civil aviation accidents are analyzed for ethanol and drugs at the Civil Aerospace Medical Institute (CAMI). Prevalence of these substances in the pilots has been evaluated on 5-yr intervals since 1989. In continuation, a fifth 5-yr study was conducted. The CAMI toxicology/medical certification and National Transportation Safety Board (NTSB) aviation accident databases were searched.During 2009-2013, samples from 1,169 pilots were analyzed for ethanol and drugs. Aircraft involved in the accidents were primarily operating under general aviation. Most airmen were private pilots and held third-class medical certificates. In relation to the first 5-yr (1989-1993) period, the pilot fatality cases decreased by 37% and the presence of ethanol and/or drugs in the pilots increased by 239% in the fifth 5-yr period. The ethanol usage was unchanged, but increases were noted with illicit and prescription drugs. The prescription drugs found were consistent with those commonly used in general population. The use of ethanol and/or drugs by aviators, along with underlying medical conditions, was determined by the NTSB to be cause/factors in 5% of the accidents. The observed decrease in the fatality cases does not necessarily suggest the decrease in aviation accidents, as the numbers of active airmen also declined.Although prevalence of ethanol and drugs has been evaluated in fatally injured aviators, such evaluation has not been performed in active pilots not involved in accidents. This type of comparative study would be crucial in assessing aviation safety.