The physical and biological parameters involved in efficient transformation of Kluyveromyces lactis by electroporation have been analyzed. By using an optimum voltage and a constant volume of cell suspension in a cuvette, the efficiency of transformation increased with increases in cell numbers and plasmid concentration. However, the most important parameter was the time of the pulse. Changes of 1 ms decreased the efficiency of transformation more than 70 to 80%. Under our best conditions, between 10 and 10 transformants per mug of plasmid DNA could be obtained. Under certain conditions, the size of the plasmid also affected electroporation efficiency. In any case, we did not obtain integrative transformation with an autonomously replicating plasmid.