A new reference cigarette, 1R6F, produced by the Kentucky Tobacco Research and Development Center, has been manufactured as a substitute for the 3R4F reference cigarette because of a depletion of 3R4F stock. The purpose of the current study was to investigate the interchangeability of 1R6F and 3R4F by comparing the chemical and biological characteristics of the mainstream smoke and to assess the inter-laboratory reproducibility by comparing the results obtained in the current study with a previous report. We analyzed 45 priority chemicals required by Health Canada for regulatory reporting and assessed the toxicological effects of cigarette smoke using