Based on the hypothesis that bacteria with minimal embryo lethality might be good candidates for vertical transmission, 103 lactose-positive Escherichia coli isolates were collected from different broiler-related conditions (sources) and analyzed using a variety of in vitro assays: biochemical profiles, sensitivity to antimicrobials, and the presence of plasmids in the 2000- to 16,000-base pair range. The results of these assays were analyzed to determine if they were associated with, or could be used as predictors of, the degree of lethality these isolates produced in 12-day-old embryos. In addition, the in vitro assay results were analyzed to determine if there was any correlation between any particular pair of factors. On the basis of biochemical profiles, the isolates were classified into 17 different groups; however, only a limited number of biochemical reactions separated a majority of the isolates. The isolates varied considerably in the number and size of plasmids they contained and in their sensitivity to the antimicrobials evaluated. The isolates also varied in their ability to kill chicken embryos—killing from 0% to 100% of those inoculated—yet significant differences were detected in lethality based on source and biochemical profile of the isolate. In addition, a predictive model for embryo lethality was constructed and evaluated based on three characteristics of these 103 isolates, namely, their ability to ferment raffinose and sorbose and their sensitivity to gentamicin.