The expression of Ca(2+)-binding protein regucalcin and its role in the regulation of protein phosphatase activity in rat brain neuronal cells obtained with primary culture was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain neuronal cells using rat regucalcin-specific primers. Moreover, regucalcin protein in brain neuronal cells was detected by Western blot analysis using a polyclonal rabbit anti-regucalcin antibody. The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward phosphotyrosine, phosphoserine and phosphothreonine in the reaction mixture containing the cytosol of neuronal cell homogenates. This increase was completely prevented by the addition of regucalcin (10(-8) M). Protein phosphatase activity toward three phosphoaminoacids was significantly elevated by the addition of Ca(2+) (100 microM) and calmodulin (5 microg/ml). This elevation was completely blocked by the addition of regucalcin (10(-8) M). The present study demonstrates that regucalcin is expressed in rat brain neuronal cells, and that it has an inhibitory effect on protein phosphatase activity in the cells.