The limulus test is a well-established method for the diagnosis of both Gram (−) sepsis and invasive fungal infection. To diagnose deep-seated fungal infections, a (1→3)-β-d-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. It is suggested that the limulus reactive substance was released from the fungi to the blood, however, its chemical properties were not precisely examined in detail because of the limited quantity available. In this study, we used chemically defined liquid medium to culture Candida spp. and collected the water soluble fraction, CAWS. The yield of CAWS was circa 100 mg/l, independent of the strain of Candida. CAWS reacted with limulus factor G (Fungitec G test MK) at concentrations as low as 100 ng/ml. Limulus factor G reactivity of CAWS was sensitive to (1→3)-β-glucanase, zymolyase and was, at least in part, bound to ConA-agarose. The ConA-bound fraction also reacted with anti-β-glucan antibody. CAWS is mainly composed of mannan and (1→6)-β-glucan, in addition to protein, assessed by ¹H-NMR spectroscopy. CAWS also reacted with typing sera of Candida spp., specific for cell wall mannan. Chemical, immunochemical and biochemical analyses of CAWS strongly suggested that the limulus factor G-activating substance was a mannan-β-glucan complex, present within the architecture of the yeast cell wall.