A cDNA microarray representing approximately 3800 cattle genes was created for functional genomic studies. The array elements were selected from > 7000 cDNA clones identified in a large-scale expressed sequence tag (EST) project that utilized spleen and normalized and subtracted placenta cDNA libraries. Sequence similarity searches of the 3820 ESTs represented on the array using BLASTN identified 3290 (86.1%) as putative human orthologs, with the remainder consisting of "novel" genes or highly divergent orthologs. Experiments were conducted with a prototype 768 gene microarray created from spleen cDNAs and with the 3800 gene array that included genes from spleen and placenta. The 768 gene array was used to profile RNA transcripts expressed by adult and fetal spleen. The 3800 gene array was used to profile transcripts expressed by adult brain and placenta. Microarray analysis of RNA extracted from fetal and adult spleen identified 29 genes that were differentially expressed two-fold or more. Transcriptional differences of two of these genes, IGJ and CTSS, were confirmed using TaqMan technology. The comparison of brain and placenta revealed 400 genes expressed at higher levels in brain and 72 genes expressed at higher levels in placenta. These results demonstrate the potential power of microarrays for understanding the molecular mechanisms of cattle development, disease resistance, nutrition, fertility and production traits.