Comparative molecular field analysis (CoMFA) has been applied to three different data sets of drug molecules binding to human rhinovirus 14 (HRV14), thermolysin and renin, respectively. Different structural alignments have been tested to predict binding properties. An alignment based on crystallographically determined coordinates of the inhibitors bound to the proteins has been compared with alignments obtained from multiple-fit and field-fit procedures. These methods are commonly used for systems where no reference to protein structural data is available. For HRV14, two different models, one based on experimental evidence and one based on a hypothetical alignment reveal moderate predictions of the binding constant of comparable quality. For thermolysin, hypothetical alignments allow a substantially better prediction than an alignment based on experimental evidence. The prediction of binding properties (expressed as delta G, delta H, and delta S) of renin inhibitors, which were aligned on the basis of crystallographic data from related inhibitors bound to the aspartyl protease endothiapepsin, gives evidence that only enthalpies (delta H) and not free enthalpies (delta G) or binding constants can be properly predicted by comparative molecular field analysis.