MOTIVATION: Spot segmentation is a critical step in microarray gene expression data analysis. Therefore, the performance of segmentation may substantially affect the results of subsequent stages of the analysis, such as the detection of differentially expressed genes. Several methods have been developed to segment microarray spots from the surrounding background. In this study, we have proposed a new approach based on Markov random field (MRF) modeling and tested its performance on simulated and real microarray images against a widely used segmentation method based on Mann-Whitney test adopted by QuantArray software (Boston, MA). Spot addressing was performed using QuantArray. We have also devised a simulation method to generate microarray images with realistic features. Such images can be used as gold standards for the purposes of testing and comparing different segmentation methods, and optimizing segmentation parameters. RESULTS: Experiments on simulated and 14 actual microarray image sets show that the proposed MRF-based segmentation method can detect spot areas and estimate spot intensities with higher accuracy.