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PURPOSE: This study uses 14 years of data from nationally representative samples of U.S. middle and high school students in the Monitoring the Future study to examine associations between school student drug testing (SDT), substance use, and participation in extracurricular activities. METHODS: Analyses use questionnaire data collected from 1998 to 2011 from 89,575 students in 883 middle schools and 157,400 students in 1,463 high schools to examine: (1) the current prevalence of SDT; (2) SDT trends over time; (3) associations between substance use and SDT type, volume, or duration among the general student population or students participating in activities subject to testing; (4) associations between students' beliefs/attitudes about marijuana use and SDT; and (5) associations between extracurricular participation rates and SDT. RESULTS: Moderately lower marijuana use was associated with any random testing of the general high school student population and for SDT of middle and high school sub-populations specifically subject to testing (athletes or participants in nonathletic extracurricular activities). However, SDT generally was associated with increased use of illicit drugs other than marijuana. CONCLUSIONS: Because the study design is observational and the data are cross-sectional, no strong causal conclusions can be drawn. However, there is evidence of lower marijuana use in the presence of SDT, and evidence of higher use of illicit drugs other than marijuana. Until further research can clarify the apparent opposing associations, schools should approach SDT with caution.