Precise analysis of the kinetics of protein/enzyme turnover in vivo has been hampered by the need to obtain multiple tissue samples at different times during the course of a continuous tracer infusion. We hypothesized that the problem could be overcome by using an overlapping (i.e., staggered) infusion of multiple stable amino acid isotopomers, which would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning over) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overlapping infusion of [2H3]leucine, [13C1]leucine, [13C1]phenylalanine, [2H5]phenylalanine, [13C6]phenylalanine, and [2H8]phenylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-to-tracee ratios of each isotopomer were measured in the plasma and jejunal free amino acid pools as well as in purified LPH polypeptides. These values were used to estimate kinetic parameters in vivo using a linear steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 +/- 1.1%/min, 17.4 +/- 11%/min, and 0.089 +/- 0.02%/min, respectively. We conclude that this multiple-tracer, single-sample protocol is a practicable approach to the in vivo measurement of protein fractional synthesis rates when only a single tissue sample can be obtained. This method has broad application and should be particularly useful for studies in humans.