The effect of insulin specific protease (ISP) in degrading plasma insulin over a wide concentration range was studied. In sera containing more than 50 muU per milliliter, more than 85 per cent of the insulin was destroyed. However, a smaller percentage of the hormone was degraded in sera containing less than 20 muU per milliliter and the results were highly variable. The residual insulin-like immunoreactivity in plasma after exposure to ISP thus represents both undegraded insulin as well as proinsulin. These findings explain the higher plasma proinsulin values obtained by the enzyme method when compared to gel filtration.