A panel of four microcell hybrids representing different sites of insertion of the exogenous neo gene into mouse chromosome 17 has been constructed. These constructions were based on a cotransfer of mouse chromosome 17 and neomycin resistance generated in a stepwise procedure involving (1) random insertion of the neo gene into a primary cell hybrid containing mouse chromosome 17 in a hamster cell background, (2) microcell-mediated chromosome transfer (MMCT) to segregate mouse and hamster chromosomes, and (3) identification of the mouse chromosome containing cells using a novel cell dotting procedure for mass screening at the cell colony level by molecular hybridization. Using this panel of four microcell hybrids for chromosome mediated gene transfer (CMGT), we obtained one transformant containing a chromosome fragment derived from the t-complex region located on mouse chromosome 17. It is concluded that the specific chromosome based procedure used here to generate CMGT transfectants may provide a general means to produce large numbers of transfectants containing megabase fragments covering, in principle, all regions of a given chromosome.