The application of a Percoll density gradient cell separation procedure to the pituitary gland of neonatal, prepubertal and mature pigs is described. After enzymatic dispersion, cell viability was 90% to 98% as determined by uptake of Trypan Blue. Recovery of dissociated cells after application of the gradient ranged from 80% to 95%. The dissociated cells were separated in several fractions that were characterized immunocytochemically using different antisera. We obtained highly enriched fractions of gonadotrope (gonadotropins), somatotrope (growth hormone) and lactotrope (prolactin) cells for each age. Concentration of the cell types (purity) was higher than 60% in the following fractions: 1) gonadotropin cells, fraction 15 (1.033 g/cc of density) from mature animals; 2) growth hormone cells, fraction 3 (1.121 g/cc of density) from neonatal animals and fraction 9 (1.087 g/cc) from prepubertal animals; and 3) prolactin cells, fraction 7 (1.094 g/cc) and 10 (1.082 g/cc) from neonatal animals and fraction 14 (1.051 g/cc) from mature pigs. In thyrotrope (thyroid-stimulating hormone) and corticotrope (adrenocorticotropin) cells, enriched fractions were also obtained, although the values of purity were lower (20% to 58%). In conclusion, the proposed cell separation and enrichment technique is suitable for the isolation, purification and examination of porcine pituitary cell types and subpopulations, and offers major advantages such as simplicity, rapidity, efficiency and reproducible results.