Computer analysis of the sequence encoding the red fluorescent protein Katushka revealed a strong donor splice site in the 3′-terminal region. A model vector encoding protein Katushka and green fluorescent protein TagGFP2 separated by a fragment of the gene tafazzin, has been constructed for experimental verification of the functional activity of this site. Splicing of this pre-mRNA should lead to the frameshift between the Katushka and TagGFP2 proteins in the case of normal splicing of the tafazzin sequence. In the case of the use of the donor splice site within a katushka sequence, it should lead to the appearance of the fusion protein Katushka-TagGFP2. Flow cytometry showed that the expression of this construct in mammalian cells led to bright red and green fluorescence. Therefore, the splice site within the gene katushka is really functional. Disruption of this splice site using site-directed mutagenesis, without changing the amino acid sequence of the Katushka protein, led to the disappearance of the green signal that corresponds to the normal splicing of tafazzin. Mutant variant of the coding sequence of the Katushka protein can be used for the analysis of pre-mRNA splicing in individual cells using fluorescence microscopy and flow cytofluorimetry. © 2011 Pleiades Publishing, Ltd.