The SO3 complexes of a few tertiary amines were tested for their ability to introduce SO3 groups into insulin. Pyridinium sulphonic acid was found to be the most suitable one. This reagent is not hydrolyzed immediately and therefore is able to exert a sulphating action for some time after being dissolved in the reaction medium. "Sulphate" groups are introduced into amino groups and, at a slower rate into tyrosine and histidine residues. By variation of the reaction conditions, insulin preparations of various sulphate content were prepared and subjected to paper electrophoresis at pH 1.7 (Fig. 3). A total number of 13 well defined, approximately equidistant bands could be observed, corresponding to insulin molecules carrying different electrical charges and convering a range from + 6 units (native insulin) to —6 units (completely sulphated insulin). The intensities of the bands in the electropherogram of a partly sulphated insulin could be related to the different reactivities of the groups concerned. The biological activity of the preparations decreased with increasing sulphate content.