DNA arrays are useful tools for simultaneously studying the expressions of a large number of genes. Herein, we describe the construction and the optimization of conditions for a low-density DNA macroarray specific for the porcine immune system. This specific DNA macroarray contains 63 gene products, including 20 cytokines, 11 chemokines, and 12 immunologically relevant receptors. It was constructed by designing gene-specific oligonucleotide primers from porcine sequences available in the EMBL or TIGR expressed sequence tag data bank and using primers from conserved regions of aligned sequences from other species for sequences unavailable for swine. Amplicons produced by reverse transcription-PCR were cloned, sequenced, and spotted onto nylon filters. A trial DNA array was first produced to optimize the intensity, specificity, and variability of signals from amplicons amplified with either gene-specific or universal primers. The DNA macroarray was then validated by comparing the gene expression profile of nonstimulated peripheral blood mononuclear cells (PBMCs) to that of phorbol 12-myristate 13-acetate and ionomycin (PMA-Iono)-stimulated PBMCs from three different animals over a 48-h time period. As already described for more conventional techniques, we showed that certain genes, such as those for CD40, gamma interferon, interleukin 2 (IL-2), the IL-2 receptor, and tumor necrosis factor alpha, were upregulated in PMA-Iono-stimulated PBMCs. A detailed analysis also indicated a downregulation of several genes which are expressed mainly by macrophages (IL-1, IL-8, AMCF-1, natural-resistance-associated macrophage protein, neutrophil chemotactic protein, DAP-12, and monocyte chemoattractant protein) in samples stimulated for 24 h with PMA-Iono compared to their levels of expression in control samples. These results indicate that the DNA macroarray that we constructed can be a useful tool for simultaneously monitoring the mRNA expression of immunologically relevant genes in different porcine samples.