In order to improve anaerobic digester productivity, rising the microbial mass in the reactor and the prediction of changes in the biomass is required. In this study the possibilities for using near-infrared spectroscopy (NIR) to monitor methanogen density in a biogas process was examined. Methane production from H-2 and CO2 was carried out with acclimated-methanogens with fed-batch substrate gas (H-2/CO2, 80:20 v/v) at pH 7 and 37 degreesC. The cells of the methanogens were washed and dried, and then original NIR spectra for predicting methanogen density were recorded. The specified absorption spectra were collected and examined. As a result, absorption spectrum peaks were found to be predominantly based on alpha proteins and lipids mainly from the cytoplasm and cell membranes of the methanogens. Furthermore, NIR was used to monitor the methane fermentation system using acetic acid as substrate. The responses from NIR analysis were cot-related to methanogen density of fermentation broth by partial least-squares regressions. The cot-relation coefficient (R), model standard error of calibration (SEC) and standard error of prediction (SEP) of the test calibration for methanogen density were 0.99, 0.14 g l(-1) and 0.55 g l(-1), respectively. For volatile fatty acids (acetic acid) R, SEC and SEP were 0.99, 0.36 g l(-1) and 0.63 gl(-1)respectively. The results indicated that within the range of the density of methanogens and the concentration of acetic acid used in this study, it was possible to monitor the important variables of methanogen density and acetate concentration simultaneously in pure substrate-fed anaerobic digesters.