A number of dramatic changes have been documented in the insulin-like growth factors (IGFs-I and -II) and their binding proteins (IGFBPs) during pregnancy. In this study we have tested the hypothesis that a failure of the normal proteolytic modification of IGFBP-3 is responsible for gestational diabetes by examining serum samples taken in the third trimester from 29 women with uncomplicated pregnancies, 21 women with established Type 1 diabetes and 20 women with gestational diabetes. Analysis of IGFBP-3 by Western immunoblotting revealed that it was present in a modified form, migrating at around 29 kDa, in the circulation of all of the women investigated. Semiquantification of the activity of the protease which modifies the IGFBP-3 demonstrated considerable variation between individuals in their ability to fragment radiolabelled IGFBP-3 following a 45-min co-incubation. Surprisingly, in one individual (with gestational diabetes) there was no detectable protease activity even though her endogenous IGFBP-3 had been modified. However, overall there was no clear-cut difference in protease activity between the different groups. Radioimmunometric analysis of IGF-I revealed significantly higher levels in women with gestational diabetes than either of the other two groups (P < 0.05). Similarly IGFBP-3 levels were also increased in these same women (P < 0.05). In contrast, IGF-II levels did not alter between the three groups. In conclusion, our hypothesis was not supported by these data and gestational diabetes was found not to be associated with any reduction in the activity of the circulating IGFBP-3 protease which could have decreased the availability of the IGF nor with any alteration in IGFs which could explain the onset of diabetes in these women.