Real time RT-PCR is the most sensitive method for quantitation of gene expression levels. The accuracy can be dependent on the mathematical model on which the quantitative methods are based. The generally accepted mathematical model assumes that amplification efficiencies are equal at the exponential phase of the reactions for the same amplicon. However, no methods are available to test the assumptions regarding amplification efficiency before one starts the real time PCR quantitation. Here we further develop and test the validity of a new mathematical model which dynamically fits real time PCR data with good correlation (R-2 = 0.9995 +/- 0.002, n = 50). The method is capable of measuring cycle-by-cycle PCR amplification efficiencies and demonstrates that these change dynamically. Validation of the method revealed the intrinsic relationship between the initial amount of gene transcript and kinetic parameters. A new quantitative method is proposed which represents a simple but accurate quantitative method. (C) 2002 Elsevier Science (USA). All rights reserved.