We have examined the ontogeny of parvalbumin (PV) and calbindin D-28k (CB) immunoreactivities in the canine anterior cingulate cortex (ACC) from the day of birth (P0) through P180. At P7, PV immunoreactivity first appeared in layer VI multipolar cells. The PV immunoreactivity in GABAergic nonpyramidal cells appeared to follow an inside-out gradient of radial emergence. Although immunoreaction was limited mainly to the developing nonpyramidal cells, pyramid-like PV immunoreactive cells were transitorily observed in layer V from P14 to P90. The developmental pattern of CB immunoreactivity differed from that of PV immunoreactivity. CB immunoreactivity first developed in layer V pyramidal cells from P0, which continued through P90. CB immunoreactive nonpyramidal cells were located in the infragranular layers and white matter at P0 and matured in both the supragranular and infragranular layers without clear inside-out gradient. This developmental study revealed the comparable belated expression of PV immunoreactivity and the transient expression of both calcium-binding proteins in layer V pyramidal cells. These results suggest that the transient expression of calcium-binding proteins in layer V pyramidal cells might be related to the critical period of early postnatal development.