Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55°C (4–18 h) and 95°C (1 h). Under these conditions at least 84–96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (≥97%) and 5-methylcytosine (≥94%) were found to be identical for both templates.