Systemic hypoxia, produced by lowering inspired PO₂, induces a rapid inflammation in several microcirculations, including cremaster muscle. Mast cell activation is a necessary element of this response. Selective reduction of cremaster microvascular PO₂ (Pmo₂) with normal systemic arterial PO₂ (PaO₂; Cremaster Hypoxia/Systemic Normoxia), however, does not elicit increased leukocyte-endothelial adherence (LEA) in cremaster venules. This could be due to a short time of leukocyte exposure to the hypoxic cremaster environment. Conversely, LEA increases when PaO₂ is lowered while cremaster Pmo₂ remains high (Cremaster Normoxia/Systemic Hypoxia). An alternative explanation of these results is that a mediator released from a central site during systemic hypoxia initiates the inflammatory cascade. We hypothesized that if this is the case, cremaster mast cells would be activated during Cremaster Normoxia/Systemic Hypoxia, but not during Cremaster Hypoxia/Systemic Normoxia. The microcirculation of rat cremaster muscles was visualized using intravital microscopy. Cremaster Pmo₂ was measured with a phosphorescence quenching method. Cremaster Hypoxia/Systemic Normoxia (Pmo₂ 7±1, PaO₂ 87±2 Torr) did not increase LEA; however, topical application of the mast cell activator c48/80 under these conditions did increase LEA. The effect of c48/80 on LEA was blocked by topical cromolyn, a mast cell stabilizer. LEA increased during Cremaster Normoxia/Systemic Hypoxia, (Pmo₂ 64±5, PaO₂ 33±2 Torr); this increase was blocked by topical cromolyn. The results suggest that mast cell stimulation occurs only when PaO₂ is reduced, independent of cremaster Pmo₂, and support the idea of a mediator which is released during systemic hypoxia and initiates the inflammatory cascade.