Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.