Cell-cell interactions are important regulatory elements in anterior pituitary (AP) physiology. As model systems to study pituitary cell-cell interactions, AP cells kept either as monolayers or as organotypic reaggregate cultures were analyzed by differential display PCR. We identified six cDNA fragments (osteopontin (Opn), connective tissue growth factor (CTGF), alpha(v)-integrin, cathepsin H, lysozyme and O-acetyl GD(3) ganglioside synthase) that showed elevated expression in monolayers compared with reaggregate cultures and the AP. The adenohypophyseal mRNA expression of Opn and CTGF, two secreted signaling substances, was studied in more detail. In situ hybridization histochemistry revealed that Opn mRNA expression is restricted to a subpopulation of gonadotropes whereas CTGF hybridization signals could not be ascribed to any known cell type. Opn transcript levels were downregulated in the APs of lactating rats and decreased when rats received s.c. injections of 17beta-estradiol for 5 days. The mRNA expression was higher in male than in female rats and increased after gonadectomy. CTGF transcript levels were higher in male compared with female rats and were increased in pregnant rats and in rats treated for 5 days with triiodothyronine or dexamethasone. These results indicate that Opn and CTGF may be of physiological importance as local communication factors in the AP.