Acylation of insulin with citraconic anhydride was studied at different pH values. The controlled acylation at pH 8.5 and 7 yielded mainly A1-citraconylinsulin (41%) and A1,B1-dicitraconylinsulin (39%), respectively. Acylation with excess reagent at pH 5.6, followed by partial deblocking at pH 5 for 30 min and 17 h, led mainly to A1,B1-dicitraconylinsulin (51%) and B1-citraconylinsulin (40%), respectively. The order of the deblocking rates for the three citraconyl groups at pH 3.5 and 5 was B29 much greater than A1 greater than B1. The biological activity of citraconyl derivatives: A1-citraconyl, B1-citraconyl and A1,B1-dicitraconylinsulin were found to be 15%, 100% and 15% (in vitro fat cell assay) and 46%, 78% and 48% (in mouse convulsion assay), respectively. In a double insulin immunoassay these derivatives had 40%, 75% and 30% immunoreactivity, respectively.