The stem-loop potential of a nucleic acid segment (expressed as a FONS value), decomposes into base composition-dependent and base order-dependent components. The latter, expressed as a FORS-D value, is derived by subtracting the value of the base composition-dependent component (FORS-M) from the FONS value. FORS-D analysis is the use of FORS-D values to estimate the potential of local base order to contribute to a stem-loop structure, and it has been used to investigate the relationship between stem-loop structure and other selective pressures on genomes. In the present study, we evaluated the reliability of FORS-D analysis by comparing it with statistically significant stem-loop potential, another robust method developed by Le and Maizel for examining stem-loop structure. We found that FORS-M values calculated using 10 randomized sequences are as reliable as those calculated using 100 randomized sequences. The resulting FORS-D values have a similar trend and distribution as statistically significant stem-loop potential, implying that FORS-D analysis is as reliable as the latter in measuring the distribution of base order-dependent stem-loop potential. Since the calculation of the FORS-M values is time consuming, the integrated program Bodslp developed by us will become a convenient tool for large-scale FORS-D analysis. The results also suggest that for some purposes the online program SigStb developed by Le and Maizel may be used as an alternative tool for FORS-D analysis.