A cDNA library was efficiently synthesized from mouse neuroblastoma poIy(A)+RNA. Several modifications of the oligo(dC)(dG) tailing procedure were used. After first strand synthesis, a dATP tail was added to the 3′-end of the cDNA. The second strand was primed for synthesis with oligo(dT). Blunt ends were produced on the cDNA by treatment with S1 nuclease. Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column. The optimal tailing time for each cDNA fraction was individually tested. Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed PstI cut pBR322.E. coliK12 RR1 cells were transformed and 2.5–5×106transformants perμg cDNA insert were obtained for each size fraction. The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive. Our modifications in the method for cDNA library synthesis had 3 advantages. (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blund ends to be tailed sychronously. This allowed a higher transformation efficiency without loss of 5′-sequences. (2)Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction. (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation. This resulted in almost 100% recovery of synthesized products at each step of this procedure.