Cyclooxygenase-2 (COX-2)-mediated prostaglandin E₂ (PGE₂) biosynthesis by macrophages (M) down-regulates microbicidal activities in innate and acquired immune responses against intracellular bacteria. Previous studies in mice showed that intraperitoneal administration of heat-killed Mycobacterium bovis bacillus Calmette-Guerin (HK-BCG) resulted in induction of splenic PGE₂-releasing M (PGE₂-M) in 7 - 14 days. In contrast, HK-BCG induced catalytically inactive COX-2 at relatively high levels in the M within 1 day. In the present study, we found that COX-2 was localized subcellularly in the nuclear envelope (NE) on days 7 and 14 after HK-BCG treatment, whereas day 1 COX-2 was dissociated from the NE. On day 1 the majority of COX-2⁺ M had phagocytosed HK-BCG. In contrast, there was no intracellular HK-BCG detected in days 7 and 14 COX-2⁺ M where COX-2 was associated with the NE. However, when M phagocytosed HK-BCG in vitro, all COX-2 was associated with the NE. Thus, the administration of HK-BCG induces the biphasic COX-2 expression in splenic M of either a NE-dissociated catalytically inactive or a NE-associated catalytically active form. The catalytically inactive COX-2⁺ M develop microbicidal activities effectively since they lack PGE₂ biosynthesis.