The relationship between gastric (GC) and tracheal (TC) colonization and the development of ventilator-associated pneumonia (VAP) remains controversial. TC, GC, and pharyngeal (PC) colonization were studied serially in 80 patients with mechanical ventilation (MV) to ascertain the routes and onset of TC. Simultaneous sample from pharynx, stomach, and trachea were obtained throughout the MV period. Quantitative cultures were performed. Seventy-two patients (90%) had TC at some time during MV. Only 19 patients presented TC after PC or GC by the same microorganisms. Indigenous gram-negative and gram-positive microorganisms colonized mainly the trachea from the start of or during MV without previous PC or GC (p < 0.05). Pseudomonas were the microorganisms causing TC principally during MV without previous PC or GC (p < 0.005). Enterobacteria produced TC without a preferential route. Of the 12 patients who developed VAP, the microorganisms responsible had already colonized the trachea in 10 patients. Only 10 of the 21 microorganisms isolated in VAP had previously colonized the pharynx or stomach. In summary, although some microorganisms have preferential routes for producing TC, the microorganisms isolated frequently change during MV. TC precedes VAP in most patients, but only a minority develop a VAP; therefore, together with TC other factors must be involved in VAP development.