Bacterial production estimates are essential in order to assess the role of bacterioplankton in the flux of carbon in the ocean. Several methods have been developed to quantify bacterial production in marine systems, but the estimation of DNA synthesis rate from measurements of the rate of tritiated thymidine incorporation has been considered the most promising since its publication in 1980. Due to its high sensitivity and apparent simplicity it has been extensively used and modified. However, our current knowledge of thymidine bacterial metabolism is still insufficient and moreover, there are some methodological issues that need to be reviewed and clarified. The accurate application of the method in natural waters requires several precautions like the extraction and purification of the DNA, the measurement of isotope dilution by extracellular and/or intracellular non-labelled thymidine, and the estimation of a conversion factor to transform thymidine incorporation into bacterial production. All these methodological requirements make the thymidine method not appropriate for routine measurements. Its application to field studies without considering these prerequisites leads to erroneous estimations of bacterial production. Implications in our understanding of the microbial ecology of marine systems are discussed.