This study was designed to evaluate the use of salivary cotinine, salivary thiocyanate, and expired-air carbon monoxide as biochemical validation measures for assessing the smoking status of adults. The participants were 20 known non-smokers plus 216 admitted smokers and 102 proclaimed quitters participating in a clinical trial of approaches to facilitate smoking cessation. Conventional analytical procedures were utilized. By use of data from known non-smokers and admitted smokers, the sensitivity and specificity of the validation measures were as follows: salivary cotinine, 99% and 100%; expired-air carbon monoxide, 96% and 100%; and salivary thiocyanate, 67% and 95%, respectively. The salivary cotinine and expired-air carbon monoxide tests confirmed smoking cessation for 55% and 74%, respectively, of the proclaimed quitters. The length of time since quitting was significantly related to the results observed with the latter measures. Consideration of these observations along with various practical factors suggests that expired-air carbon monoxide assays may be the validation measure of choice for most clinical trials.