Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A₂ (PLA₂), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA₂ and the GXIIB PLA₂-like protein. Antibodies were raised in rabbits against recombinant human PLA₂ proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 μg/L for the TR-FIA for GIB PLA₂, 1 μg/L (GIIA), 35 μg/L (GIID), 3 μg/L (GIIE), 4 μg/L (GIIF), 14 μg/L (GIII), 11 μg/L (GV), 2 μg/L (GX), 92 μg/L (GXIIA) and 242 μg/L (GXIIB). All secreted PLA₂s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA₂ and the GXIIB PLA₂-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA₂ in serum (median 19.0 μg/L, range 13.1–33.7 μg/L, n=4) as compared to the healthy blood donors (median 1.8 μg/L, range 0.8–3.4 μg/L, n=28, P<0.0001). The concentration of GIIA PLA₂ in the sera of septic patients (median 315.7 μg/L, range 15.9–979.6 μg/L, n=34) was highly elevated as compared to that of the blood donors (median 1.8 μg/L, range 0.8–5.8 μg/L, n=28, P<0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA₂ in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA₂s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA₂ and the GXIIB PLA₂-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA₂s above the detection limits of the current TR-FIAs.