A solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for the determination of human complement3 (C3) based on a sandwich type assay and a labelled avidin-biotin (LAB) type assay was described. The anti-human complement3 and avidin were labeled with eosin5-isothiocyanate. On a polyamide membrane (PM), SS-RTP signals (lambda(ex)/lambda(em) = 535/678 nm) of immune complexes obtained by both assays were linear with the concentration of complement3 in the range of 6.25-100 ng/ml. The detection limits are 1.37 ng/ml for sandwich assay and 2.74 ng/ml for labeled avidin-biotin assay. (For a sample volume of 0.4 microl per spot, the mass detection limits are 0.546 and 1.09 pg/spot, respectively. If the molecular weight of human complement3 is 185,000, the detection limits are 2.95 and 5.91 amol per spot.) The results of determination of complement3 in 20 human sera obtained by labeled avidin-biotin SS-RTP-IA are correlated well with those obtained by ELISA. This study shows that SS-RTP-IA by whichever direct, sandwich or labeled avidin-biotin type assay can combine very well the characteristics of both the high sensitivity of SS-RTP and specificity of the immunoreaction.