RNA secondary structure plays critical roles in several biological processes. For example, many trans-acting noncoding RNA genes and cis-acting RNA regulatory elements present functional motifs, conserved both in structure and sequence, that can be hardly detected by primary sequence analysis alone. We describe here how conserved secondary structure motifs shared by functionally related RNA sequences can be detected through the software tool RNAProfile. RNAProfile takes as input a set of unaligned RNA sequences expected to share a common motif, and outputs the regions that are most conserved throughout the sequences, according to a similarity measure that takes into account both the sequence of the regions and the secondary structure they can form according to base-pairing and thermodynamic rules. The method is split into two parts. First, it identifies candidate regions within the input sequences, and associates with each region a locally optimal secondary structure. Then, it compares candidate regions to one another, both at sequence and structure level, and builds motifs exploring the search space through a greedy heuristic. We provide a detailed guide to the different parameters that can be employed, and usage examples showing the different software capabilities.