A strategy for PCR-amplification and sequencing of the flanking regions in the polymorphism D2S44 (YNH24) has been developed based on the investigations of Edwards et al. (1991). The flanking regions of the YNH24 probe were successfully amplified and two distinct PCR products with fragment sizes of 180 and 250 bp obtained. After asymmetric PCR and didesoxy-sequencing 60 bp could be determined for every PCR fragment. D2S44-specific primers were constructed which were located at the transition between the flanking and repeat regions. Amplification conditions were optimized using the YNH24 probe, different nuclease S1 concentrations and incubation times. Optimized conditions were applied to the amplification assay of human D2S44 alleles which had been investigated by RFLP analysis.