The construction of cDNA libraries is useful in understanding gene expression in organisms under different conditions, but random sequencing of unbiased cDNAs is laborious and gives rise to redundancy. We aimed to isolate cDNAs induced by switching Aspergillus nidulans from glucose to selected polysaccharides. We devised a Negative Subtraction Hybridization method which involved screening a library made from cDNAs from cells grown under a selected physiological condition with labeled cDNA probes from another physiological condition. In a screen for transcripts induced by switching the carbon source, 3,532 negatives were isolated. Single-channel microarray experiments were done to confirm that the majority of the negatives were indeed induced differentially. To gain insight into the regulation, we used microarrays to identify gene expression changes following a shift from glucose to pectin or glucose starvation. The findings from this study portray the useful utilization of the powerful microarray technology to study gene expression profiling under various physiological conditions.