In the Croatian transfusion medicine, no general agreement has yet been achieved whether red blood cell (RBC) Rhesus (Rh) antibodies detected during pregnancy only by enzyme tests can cause hemolytic disease of the newborn (HDN). Results of the detection of clinically significant RBC antibodies by low-ionic-strength additive solution antiglobulin test (LISS-IAT) and trypsin enzyme test in 22,947 pregnant women are presented. All pregnant women in whom clinically significant RBC antibodies (RBC-CSA) were detected by LISS-IAT and/or enzyme tests were followed and observed during pregnancy. The women who had enzyme-only anti-D antibodies in their serum were followed up during subsequent pregnancies. Out of 302 positive results obtained by both techniques, irregular clinically significant enzyme-only antibodies (anti-RhD and anti-RhE specificity) were detected in 14 (4.6%) pregnant women. None of 11 RhD positive newborns whose mothers had enzyme-only anti-D antibodies, had signs of HDN after delivery. In these 11 women, anti-D antibodies were detected by LISS-IAT in the first trimenon of subsequent pregnancy. Nine infants born from subsequent pregnancies to women who had previously had enzyme-only anti-D, had clinical signs of HDN. The authors concluded that there is no need for enzyme tests in prenatal testing because enzyme tests are not reliable in the prediction of HDN.