A number of different tools have been made available for the automated analysis of T-RFLP data, all with their own merits. T-Align [10] can be used for alignment of duplicates, generation of consensus profiles and alignment of the consensus profiles. However, the number of replicates is fixed to two, and there are no options for normalization. T-REX [17] has two options for alignment: either simply replacing the observed T-RF sizes with the value of the nearest integer or using an adaptation of the T-Align alignment method. However, there is only one option for noise removal, the method by Abdo et al. [7] and there is no flexibility in the generation of consensus profiles. The T-RFLP Stats scripts by Abdo et al. [7] only perform the methods described in the paper without any other options. None of the available tools enable easy comparison of different normalization strategies. They also lack options for consensus profile generation and tools for the evaluation of the T-RF alignment. To meet this need, the collection of Visual Basic macros and the analysis template presented here, from now on referred to as the Tools for T-RFLP data analysis, were developed. The procedures allow for automated normalization and alignment of replicate profiles, creation of consensus profiles from replicate profiles and normalization and alignment of the consensus profiles. Included are also procedures to evaluate the accuracy of the resulting alignments, to calculate association coefficients between T-RF profiles and a diversity index. The macros and the analysis template sheet are designed so that adjustments of the parameters for noise baseline, T-RF analysis range, normalization and alignment can easily be made. In addition, all analyses can be based on either peak height data or peak area data.