A proper evaluation of the physiological significance of plasma prorenin depends on its accurate determination. However, current activation methods do not necessarily measure total prorenin, or a known proportion of it, even when carried out to apparent completion. Thus, extending cold activation of human plasma at -4 degrees C generally revealed progressive increments of prorenin, mainly during the first 15 days, but the total and the time required to achieve it varied considerably among individuals. Similarly, the titration curves of individual plasmas varied with increments of added trypsin and achieved totals that were not necessarily greater than those obtained by cold activation. This indicates the inappropriateness of attributing greater effectiveness to one method over the other. When the two methods were paired in sequence, a synergism was apparent in that prorenin estimates increased consistently; in one case more than 10-fold. Thus, total prorenin by any single method generally fell short of the total achieved by double methods. However, this too may still not represent the unknown true total prorenin. The sequence of activation steps was important, providing clues as to the mechanism of the observed synergism. Trypsin-before-cold activation proved to be more effective than trypsin-after-cold activation, with no further advantage being gained from triple treatments involving cold before and after trypsin, or trypsin before and after cold. The inferiority of trypsin-after-cold activation was apparently due to sensitization of the plasma to the destructive effects of trypsin, shifting its titration curve to the left.(ABSTRACT TRUNCATED AT 250 WORDS)