Cellular metabolism studies were performed comparing human insulin with two rapid-acting analogs, LysB28ProB29 insulin (LysPro) and AspB10 insulin (B10-Asp). B10-Asp bound to isolated hepatocytes at 37 degrees C to a greater extent than LysPro or native insulin, which were equivalent. The rate of degradation was similar for the three materials, resulting in a significant reduction in the degraded/bound ratio for the B10 analog. The processing of membrane-bound material was examined by incubating cells with hormone at 4 degrees C, removing unbound insulin, and incubating the cells at 37 degrees C. Again, binding was greater for B10-Asp versus LysPro or native insulin, with a reduction in the degraded/bound ratio. Hormone internalization and processing was examined by an acid wash of cells incubated with 125I(A14)-labeled hormone to remove surface-bound materials. The processing rate was slower for B10-Asp versus LysPro or native insulin. Cell extraction and examination on molecular-sieve chromatography confirmed that B10-Asp was processed at a slower rate than either LysPro or native insulin. Intact B10-Asp was found in the cell after 4 hours, whereas all native insulin and LysPro were degraded by 90 to 120 minutes. B10-Asp also caused a greater incorporation of thymidine into DNA in cultured cells than LysPro or native insulin, which were similar. These data show that the cellular processing of LysPro is essentially identical to that of native insulin. However, B10-Asp has markedly different properties and is processed much more slowly. The prolonged cell residence time of B10-Asp could contribute to its greater effects on cell growth and mitogenesis.