We have extended the previous "Benchmark RNA Alignment dataBase" BRAliBase II by a factor of 30 in terms of the alignment number and with respect to the range of sequences per alignment. With the new datasets of BRAliBase 2.1 we tested several sequence alignment programs. Obviously it is not possible to test all available programs; here we concentrated on well-known sequence alignment programs and those already identified as good aligners in our first study [22]. Additionally we showed that gap-parameters can be (easily) optimized and tested whether the incorporation of RNA-specific substitution matrices results in a performance change.