Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.