Colony morphology and the spot-indole test were evaluated to assess the practicality of the rapid identification of Escherichia coli isolates directly from primary isolation plates. One hundred ninety-one lactose-fermenting, gram-negative bacilli were identified by standard testing procedures (either API-20E, Micro-ID, or a 5-test biochemical battery). These isolates also were tested with the spot-indole test, using either a 1% or 5% p-dimethylaminobenzaldehyde or 1% p-dimethylaminocinnamaldehyde reagent. Of the total 191 E. coli isolated, 128 (67.0%) were single-colony types on the primary plates, and 112 (58.6%) met the morphologic criteria and were positive and spot-indole testing. Of these 112, 111 (99.1%) were identified accurately as E. coli by the morphologic criteria/spot-indole approach. This method can be recommended as a practical diagnostic aid in the clinical microbiology laboratory as it allows significant cost and time savings for the majority of the most frequently isolated gram-negative bacilli.