AbstractNative culture fluorescence was investigated as an additional source of information for predicting biomass and glucose concentrations in a fed‐batch fermentation of Alcaligenes eutrophus. Partial least squares (PLS) regression and a feed forward neural network (FFNN) coupled with principle component analysis (PCA) were each used to model the kinetics of the fermentation. Data from three fermentations was combined to form a training set for model calibration and data from a fourth fermentation was used as the testing set. The fluorescent soft‐sensors were compared with a previously developed feed forward neural network soft‐sensor model which used oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), aeration rate, feed rate, and fermentor volume to estimate biomass and glucose concentrations. The best model performance for predicting both biomass and glucose concentrations was achieved using the native fluorescence‐based models. Real data predictions of the biomass concentration in the testing set were obtained using both the PLS and FFNN PCA modeling utilizing fluorescence measurements plus the rate of change of the fluorescence measurements. Accurate predictions of the glucose concentration in the testing set were obtained using the FFNN PCA modeling technique utilizing the rate of change of the fluorescence measurements. Substrate exhaustion was indicated qualitatively by a first‐order PLS model utilizing the rate of change of fluorescence measurements. These results indicate that native culture fluorescence shows promise for providing additional valuable information to enhance predictive modeling which cannot be extracted from other easily acquired measurements. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 104–111, 2003.