A cDNA library was generated from rat brain tissues and organized into 1536-well plates, using a fluorescence activated cell sorter (FACS), acting as a single cell deposition system. The organized library containing 10 000 clones, with 60% full-length cDNA inserts, allowed the generation of multiple identical membrane replicas. Each replica was hybridized with a complex probe obtained from a particular brain tissue or a given cultured cell. The signal intensity for each of the clones present on the membrane, quantified with a standard image-analysis software, is proportional both to the abundance of the corresponding mRNA in the probe and to the amount of plasmid template on the membrane. The latter value was thus used to normalize the signals produced with complex probes, to optimize the comparison of mRNA expression levels for the different systems under study. The construction of high-quality cDNA libraries, the generation of identical membrane replicas and comparable probes, and the utilization of an image-analysis software package, coupled with the normalization of the spot intensity by assaying plasmid quantity, significantly improves the differential screening approach. Altogether, these technical improvements open the possibility to compare a great number of different probes and, in consequence, to accumulate biological information for each clone present in an organized cDNA library. The functional information obtained should complement data from DNA sequencing projects.