Microarray technology permits high-throughput comparisons of gene expression in different parasite stages or sexes and has been used widely. We report the first use of this technology for analysis of gene expression in filarial male and female worms. The slide array (comprised of 65-mer oligos representing 3569 EST clusters) was spotted with sequences selected from the extensive Brugia malayi EST database (). Arrays were hybridized with Cy dye labeled male and female cDNA. The experimental design included both biological and technical (dye-flip) replicates. The data were normalized for background and probe intensity, and the relative abundance of hybridized cDNA for each spot was determined. Genes showing two-fold or greater differences with P<0.05 were considered gender-regulated candidates. One thousand one hundred and seventy of 2443 clusters (48%) with signals above threshold in at least one sex were considered as gender-regulated gene candidates. This included 520 and 650 clusters up-regulated in male and female worms, respectively. Fifty of 53 (94%) gender-regulated candidate genes identified by microarray analysis were confirmed by real-time RT-PCR. Approximately 61% of gender-regulated genes had significant similarity to known genes in other organisms such as Caenorhabditis elegans. Many C. elegans homologues of these genes have been reported to have reproductive phenotypes (sterility or abnormal embryo development) by RNA interference. This study has provided the first broad view of gender-regulated gene expression in B. malayi; this should lead to improved understanding of reproduction in filarial nematodes. More generally, this approach holds great promise as a means of studying stage-specific or tissue-specific gene expression in parasitic nematodes.