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Lymphocytes from peripheral blood of carp proliferate in a clonal culture in soft agar, in the presence of phytohemagglutinin, generating several morphologically distinct types of colonies. Cells from colonies developing on the surface of the agar (surface colonies) and cells from colonies developing within the agar (agar colonies) were studied. Several differences were found between cells from the two types of colonies with respect to morphology, ultrastructure and the distribution of cytoplasmic determinants antigenically related to serum immunoglobulin. Colonies were quantitated as a function of the number of cells seeded, in primary cultures of peripheral blood leukocytes and in secondary (replated) cultures of isolated surface colony cells. The numbers of surface colonies and agar colonies in the two systems were comparable. Preferential formation of surface over agar colonies was noted, and there was an initial concentration of cells (individual for each fish) which resulted in optimal colony growth. This method was found to be suitable for isolating highly homogeneous subpopulations of PHA-responsive lymphocytes, which could subsequently be further expanded in liquid culture. A requirement for an exogenously produced growth factor (possibly similar to mammalian Interleukin 2) in the maintenance of long-term clonal cultures is suggested.