The described procedure is a simple and rapid method for isolating pure lymphocytes from carp gut epithelium. The advantage of the described technique of i-IELs extraction is that it allows for the extraction of the cells from each carp, making it possible to study their characteristics on an individual basis in fish. In this procedure, gut intraepithelial lymphocytes are separated from the epithelium layer and other lymphoid cells by Percoll gradient centrifugation. Since this procedure results in high recovery and purity in a preparation of i-IELs , there is no need for multiple nylon wool filtrations. The obtained i-IELs population could be utilised for molecular, biochemical or phenotypic characterisation, as well as for functional evaluation, such as proliferative and cytotoxic activities.