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The objective of this paper was to investigate the segmentation ability of the fuzzy Gaussian mixture model (FGMM) clustering algorithm, applied on complementary DNA (cDNA) images. Following a standard established procedure, a simulated microarray image of 1600 cells, each containing one spot, was produced. For further evaluation of the algorithm, three real microarray images were also used, each containing 6400 spots. For the task of locating spot borders and surrounding background (BG) in each cell, an automatic gridding process was developed and applied on microarray images. The FGMM and the Gaussian mixture model (GMM) algorithms were applied to each cell with the purpose of discriminating foreground (FG) from BG. The segmentation abilities of both algorithms were evaluated by means of the segmentation matching factor, coefficient of determination, and concordance correlation, in respect to the actual classes (FG-BG pixels) of the simulated spots. Pairwise correlation and mean absolute error of the real images among replicates were also calculated. The FGMM was found to perform better and with equal processing time, as compared to the GMM, rendering the FGMM algorithm an efficient alternative for segmenting cDNA microarray images.