In the present study, three luminol signal enhancers {4-methoxyphenol, 4-hydroxybiphenyl and 4-(1H-pyrrol-1-yl)phenol} were utilized in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP). The latter was applied in a heterogenous enzyme immunoassay that has been previously described. The employment of these molecules greatly affected important assay parameters, such as detection limit and the range of the calibration curve and the results were compared with those obtained from other two similar enhancers that have been described from our group. Practically, the use of a novel enhancer, even if this is a slightly changed 4-substituted phenol derivative, can affect assay properties so dramatically, one can assume that another substrate/enzyme system was applied. Furthermore, the use of different luminol signal enhancers in the luminol/HRP/H(2)O(2) system affected not only the intensity of the obtained signal, which is well known, but also its kinetics. It was monitored that the stronger intensity was combined with a more rapid decrease of the CL signal.