Cannabis is the most commonly consumed illicit drug worldwide. As few studies characterized differences between occasional and frequent cannabis smokers, we developed a controlled cannabis administration study to investigate pharmacodynamic and pharmacokinetic differences between these groups. Eleven occasional and 14 frequent smokers smoked one 6.8% delta9-tetrahydrocannabinol (THC) cannabis cigarette ad libitum over 10 min. Psychomotor, working memory, risk-taking, subjective, and physiological effects were monitored, and blood, plasma, oral fluid (OF), urine, and breath collected for up to 30 h after smoking. Optimal storage conditions for urinary cannabinoid stability were defined. Draeger DrugTest 5000 onsite OF testing device performance was determined, and a new OF quantification assay for six cannabinoids was developed. Occasional cannabis smokers had increased psychomotor tracking errors and impaired divided attention and reaction time after controlled cannabis smoking, while frequent smokers demonstrated partial tolerance to cannabis' psychomotor effects. Occasional smokers reported longer and more intense subjective effects compared to frequent smokers. Frequent smokers had higher blood and plasma cannabinoid concentrations, even after accounting for residual baseline concentrations. In OF, frequent smokers had significantly longer windows of cannabinoid detection with various screening and confirmation cutoffs. The DrugTest 5000's diagnostic sensitivity, specificity, and efficiency with a 5-µg/L screening cutoff and THC confirmation at 2 µg/L with Quantisal, Statsure and Oral-Eze OF collection devices were 74.0-90.7, 75.0-94.1, and 80.0-87.9%, respectively. Performance varied based on the different analytes included in the confirmation analysis. Frequent smokers had significantly higher urinary THC-glucuronide, 11-nor-9-carboxy-THC (THCCOOH), THCCOOH-glucuronide, and total THCCOOH concentrations than occasional smokers after smoking cannabis, even after normalizing for urinary creatinine concentrations. Frozen storage provided optimal cannabinoid stability in urine, with THC-glucuronide and THCCOOH-glucuronide being stable for up to 6 months. Our new validated method for the analysis of cannabinoids in OF is the most comprehensive method developed to date. Limits of quantification (LOQ) were 15 ng/L for THCCOOH and 0.2 µg/L for THC, 11-hydroxy-THC, tetrahydrocannabivarin, cannabidiol, and cannabigerol. Our data will aid scientists in cannabinoids identification and interpretation in clinical, driving under the influence of cannabis, criminal justice, anti-doping, and workplace drug testing settings and in the development of evidence-based drug policy.