Multiple sequence alignment is an essential tool in many areas of biological research, and the accuracy of an alignment can strongly affect the accuracy of a downstream application such as phylogenetic analysis, identification of functional motifs, or polymerase chain reaction primer design. The heads or tails (HoT) method (Landan G, Graur D. 2007. Heads or tails: a simple reliability check for multiple sequence alignments. Mol Biol Evol. 24:1380-1383.) assesses the consistency of an alignment by comparing the alignment of a set of sequences with the alignment of the same set of sequences written in reverse order. This study shows that HoT scores and the alignment accuracies are positively correlated, so alignments with higher HoT scores are preferable. However, HoT scores are overestimates of alignment accuracy in general, with the extent of overestimation depending on the method used for multiple sequence alignment.