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A chromogenic agar, R&F Enterobacter sakazakii chromogenic plating medium (ESPM), was developed for isolating presumptive colonies of E. sakazakii from foods and environmental sources. ESPM contains two chromogenic substrates (5-bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D-cellobioside), three sugars (sorbitol, D-arabitol, and adonitol), a pH indicator, and inhibitors (bile salts, vancomycin, and cefsulodin), which all contribute to its selectivity and differential properties. On ESPM, 79 pure culture strains of E. sakazakii (10 clinical isolates and others from food and environmental sources) yielded blue-black (three strains were blue-gray) raised colonies, 1 to 2 mm in diameter with and without halos after 24 h at 35 degrees C. Other enteric organisms plus Pseudomonas aeruginosa yielded white, yellow, green, or clear colonies with and without clear halos. Of these genera, only Shigella sonnei and one Pantoea strain produced blue-black to blue-gray colonies. ESPM was used to isolate E. sakazakii from a variety of foods: corn, wheat, and rice flours; powdered infant formula; dairy products (dried milk, whey, and caseinates); cereals; and environmental sources. Most false-positive results on ESPM were eliminated by observing acid production on either sucrose or melibiose after 6 h at 35 degrees C on a R&F E. sakazakii screening medium (ESSM) biplate. In an analysis of 240 samples, the number of samples positive for E. sakazakii by the ESPM-ESSM method and the U.S. Food and Drug Administration protocols (violet red bile glucose agar and tryptic soy agar) were 27 and 16, respectively, with sensitivity and specificity values of 100.0 and 96.9% versus 59.3 and 43.7%, respectively. These data support the fact that E. sakazakii confirmation should be based on more than one confirmation system. Both the API 20E and Biolog Microlog3 4.20 systems should be used for confirmation of E. sakazakii isolates.