In both mice and humans, a subset of platelets can be identified that shows increased labeling with nucleic acid-specific fluorescent dyes, such as thiazole orange. Termed" reticulated platelets," they have been postulated to be platelets that have recently entered the circulation. Their numbers appear to reflect the rate of new platelet production in a number of clinical and experimental situations. To determine whether reticulated platelets really are the youngest platelets in circulation and to estimate the length of time that they are identifiable after entering the circulation, we have employed a technique of" in vivo biotinylation" in mice that labels the entire cohort of circulating cells with covalently bound biotin. Blood samples can then be double-labeled with fluorescent avidin derivatives and thiazole orange, permitting correlated measurement of both surface biotin content and nucleic acid content. The biotinylation occurs rapidly, is complete within 30 minutes, is stable for several days, and does not appear to alter platelet function. The results show that within 24 hours after in vivo biotinylation, platelets appear in the circulation with decreased levels of biotinylation and that these are the reticulated platelets. The estimated lifespan of reticulated platelets is 1.8 days, and the lifespan of all platelets by this method is 4.5 days, which is in agreement with estimates made by other methods.