Whole-genome profiling of gene expression in a segregating population has the potential to identify the regulatory consequences of natural allelic variation. Costs of such studies are high and require that resources--microarrays and population--are used as efficiently as possible. We show that current studies can be improved significantly by a new design for two-color microarrays. Our "distant pair design" profiles twice as many individuals as there are arrays, cohybridizes individuals with dissimilar genomes, gives more weight to known regulatory loci if wished, and therewith maximizes the power for decomposing expression variation into regulatory factors. It can also exploit a large population (larger than twice the number of available microarrays) as a useful resource to select the most dissimilar pairs of individuals from. Our approach identifies more regulatory factors than alternative strategies do in computer simulations for realistic genome sizes, and similar promising results are obtained in an application on Arabidopsis thaliana. Our results will aid the design and analysis of future studies on gene expression and will help to shed more light on gene regulatory networks.