Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.