By taking advantage of the sequence homology of currently available EF-hand loops and the flanking structural contents, we generated a series of patterns for the prediction of EFhand proteins. We have modified the pattern PS00018 by allowing more choices (Glu, Gln, and Ser) at position 1 and adding constraints at the flanking helical regions for canonical EF-hand motifs. In addition, several patterns have been developed to identify EF-hand like sites with different structural elements flanking the loop. Further, to circumvent the problem of identifying the pseudo EF-hand loop, a pattern has been developed by moderately loosening the constraints at the paired C-terminal canonical EF-hand and incorporating reserved residues in the N-terminal pseudo EF-hand. Compared with the original pattern PS00303, the new pattern reflects conserved genomic information in both EF-motifs and significantly improved the predictive accuracy and sensitivity [41] .