We have developed a method to monitor mRNA expression that is based upon the reverse transcriptasepolymerase chain reaction (RT-PCR) and includes multiple sets of primer pairs in complification reactions. To observe relative changes in mRNA steadystate levels, each target in a multiplex reaction was amplified to within a predetermined range by using PCR cycle numbers specific for each target. Optimal PCR cycle numbers for target templates were determined by preliminary titration experiments performed using the "primer-dropping" method. By employing this method, the overall amplification reaction was limited, permitting the PCR products to remain within the exponential range of the amplification curve and yet be detectable on ethidium bromide-stained gels. We demonstrated the utility of this method by monitoring the expression kinetics of cyclins (A, B 1, D 1), and (E), and of the immediate-early genes (\mathbf{c})-fos, (\mathbf{c})-myc, and (\beta)-actin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the multiplex reactions as an endogenous internal standard to control for variations in product abundances due to differences in individual RT and (P C R) reaction efficiencies. Changes in gene expression of less than twofold to greater than 75 -fold were readily distinguished. 1994 Academic Press, Inc.