Understanding transcriptional regulation is a major goal of molecular biology. Motif expression decomposition (MED) was recently introduced to describe the expression level of a gene as the sum of the products of the binding strengths of its cis-regulatory motifs and the activities of the corresponding trans-acting transcription factors (TFs). Here, we use computer simulation to examine the accuracy of MED. We found that although MED accurately rebuilds gene expression levels from decomposed motif binding strengths and TF activities, estimates of motif binding strengths and TF activities are unreliable. Nonetheless, MED provides accurate estimates of relative binding strengths of the same motif in different genes and relative activities of the same TF under different conditions. We found that reasonably accurate results are achievable with genome-wide expression data from only 30 conditions and that MED results are robust to the existence of unknown occurrences of known motifs, although they are less robust to the presence of unknown motifs. With these understandings, judicious use of MED will likely provide useful information about eukaryotic transcriptional regulation. As an example, MED results are used to demonstrate that motifs generally have higher binding strengths when appearing in multiple copies than appearing in one copy per promoter.