We recently provided evidence that anti-inflammatory macrophage activation, i.e. the inhibition of constitutive and signal-induced NF-B activity by the pulmonary collectin surfactant protein (SP)-A, critically involves a promoted stabilization of inhibitory B (IB)-, the predominant inhibitor of NF-B, via posttranscriptional mechanisms comprising the activation of atypical (a) PKC. SP-A uptake and degradation by alveolar macrophages (AM) occur in a receptor-mediated, clathrin-dependent manner. However, a mutual link between endocytosis of and signaling by SP-A remains elusive. The aim of this study was to investigate whether clathrin-mediated endocytosis (CME) of SP-A by AM is a prerequisite for its modulation of the IB-/NF-B pathway. The inhibition of clathrin-coated pit (CCP) formation and clathrin-coated vesicle (CCV) formation/budding abrogates SP-A-mediated IB- stabilization and SP-A-mediated inhibition of LPS-induced NF-B activation in freshly isolated rat AM, as determined by Western analysis, fluorescence-activated cell sorting, confocal microscopy, and EMSA. Actin depolymerization and inhibition of CCP formation further abolished SP-A-mediated inhibition of LPS-induced TNF- release, as determined by ELISA. In addition, SP-A-induced atypical PKC activation was abolished by pretreatment of AM with CCV inhibitors as determined by in vitro immunocomplex kinase assay. While CME is classically considered as a means to terminate signaling, our results demonstrate that SP-A uptake via CME by AM has to precede the initiation of SP-A signaling.