1. The ability of calcimycin, cyclopiazonic acid and thapsigargin to facilitate the N-methyl-D-aspartate (NMDA)-mediated depolarization of cortical projection neurones was investigated by use of grease-gap recording and the results compared with the facilitation that results from activation of 5-hydroxytryptamine2A receptors. 2. Calcimycin (0.25 to 3 microM), cyclopiazonic acid (5 to 30 microM), and thapsigargin (10 to 300 nM) reversibly facilitated the NMDA (50 microM)-induced depolarization in the presence of tetrodotoxin. The concentration-response relationships were bell-shaped with a mean enhancement of 550% for calcimycin (1 microM) and approximately 400% for cyclopiazonic acid (20 microM) and thapsigargin (100 nM). At the highest concentration of each agent tested, no facilitation was observed. 3. Chlorpromazine (1 microM) partially restored a facilitation at 3 microM calcimycin and 300 nM thapsigargin. Myo-inositol (10 mM) and 100 nM staurosporine were both ineffective in this regard. 4. The depolarization elicited by 10 microM quisqualate or 5 microM kainate was not facilitated by 10 microM cyclopiazonic acid. 5. Calcimycin (0.5 microM), cyclopiazonic acid (20 microM), and thapsigargin (100 nM) elicited a significant facilitation in the presence of an antagonist cocktail consisting of D,L-2-amino-3-phosphonopropionic acid, prazosin, ritanserin, and scopolamine, although the magnitude of the facilitation was reduced. 6. Facilitation of the NMDA depolarization elicited by both 30 microM 5-hydroxytryptamine and 10 microM phenylephrine was eliminated in nominally Mg(2+)-free medium. In contrast, the facilitation induced by 0.5 microM calcimycin remained intact. 7. Bis-(o-aminophenoxy)-ethane-N,N,N,N, tetraacetic acid aminoethoxy (50 microM) or perfusion with nominally Ca(2+)-free medium eliminated facilitation of the NMDA depolarization induced by 30 microM 5-hydroxytryptamine and 100 nM thapsigargin. 8. The facilitation induced by both 30 microM 5-hydroxytryptamine and 1 microM calcimycin was reduced in a concentration-dependent manner by nifedipine (1 to 10 microM). 9. Calcimycin, cyclopiazonic acid and thapsigargin facilitate the NMDA depolarization in a manner which closely mimics the facilitation induced by 5-hydroxytryptamine. It is concluded that enhancement of the NMDA depolarization at cortical projection neurones results from an elevation of Ca2+ in the cytosol and that several sources of Ca2+ contribute to the facilitation.