Kentucky Tobacco Research & Development Center reference cigarettes are widely used in tobacco research laboratories as tobacco reference products in various areas including analytical method development and modified risk tobacco product development. Reference cigarettes provide stable physical and chemical characteristics for these investigations. Storage conditions are critical for maintaining reference cigarette quality within its effective period. Some experimental data from the literature indicate our current storage conditions are adequate to store our reference cigarettes. However, there is no integrated data to support this conclusion. The objective of this study was to analyze filler tobacco contents and smoke from 3R4F cigarettes after 1, 2, 3, 6, 9 and 12 months of storage at -20°C, 4°C and room temperature to determine stability of selected constituents. The sampled cigarettes were conditioned for 48hr at 22°C and 60% relative humidity prior to filler analysis and smoking. Filler analysis included oven volatiles, individual alkaloids and TSNAs. Smoke analysis included measurement of individual alkaloids and TSNAs, CO and TPM under ISO smoking regime. Five replications of 20 cigarettes in a single smoking machine run (five cigarettes per port) were assayed. There were no significant changes in the oven volatiles, nicotine and NNN in the filler from the three storage conditions. Also, there were no significant changes in puff/cigarette, CO, TPM, alkaloids and TSNAs in the smoke of cigarettes stored in these conditions. Our experimental data demonstrated 3R4F cigarettes were relatively stable after one year storage under these storage conditions.