We use modified oligonucleotides with enhanced strength of complementary DNA binding for primer walking DNA sequencing with strings of short contiguous oligonucleotides as primers. Such an approach allows us to reduce the probability of primer failures due to unstable binding of oligos with templates. In this paper the factors affecting the priming efficiency of segmented primers (strings composed of several short oligonucleotides contiguously juxtaposed on the template) used for DNA sequencing were investigated. Modified oligonucleotides were used to discriminate the effects caused by intrinsic properties of the oligonucleotides and by template features. It was shown that the most crucial factor is the stability of the duplex formed by the template with the 3′-outermost oligonucleotide of a string. The data were obtained with a model M13 template and in the process of sequencing the region flanking a long terminal repeat of human endogenous retrovirus HERVK mapped on chromosome 19. The sequencing was done by primer walking with strings of contiguous modified hexanucleotides. The effects revealed should be taken into consideration when choosing oligonucleotide units of segmented primers and for construction of minimised libraries composed of short unit oligonucleotides. © 1996 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.