Data on the frequency of anti-platelet factor 4/heparin (PF4/H) antibodies and their association with outcomes in intensive care unit (ICU) patients are sparse. In this prospective, observational study we screened 320 consecutive surgical/medical ICU patients for anti-PF4/H antibodies by enzyme-immunoassay (EIA) for immunoglobulin (Ig)G/A/M separately and heparin-induced platelet activation assay (HIPA) at ICU admission (=baseline), day 6, and day 10. HIPA-positive patients were additionally tested by serotonin-release assay (SRA). Patients tested positive by day 10: for anti-PF4/H-IgG = 17.2 % and for anti-PF4/H-IgM = 42.1 %. Within the first 10 ICU days, platelet counts decreased to <100 Gpt/L in 27.8 % patients. However, only seven patients (2.2 %) experienced a drop in the platelet count ≥50 % beginning after the fourth ICU day. These included the only two patients (0.6 %; 95 % confidence interval 0.08-2.2 %) with heparin-induced thrombocytopenia (HIT). Only strong reactions in the HIPA were reproducible by SRA. This study confirms that testing for anti-PF4/H IgG antibodies should be restricted to ICU-patients who develop a platelet count decrease of >50 % that begins after day four of heparin treatment (which may have started before ICU admission). Among patients testing positive by IgG-specific EIA a functional platelet activation assay should be performed (regarding only strong reactions as positive).