Cholera toxin subunit B (CTB) is a sensitive neuroanatomical tracer that generally transports retrogradely in the nervous system, and has been used extensively in brightfield microscopy. Recently, Alexa Fluor (AF) conjugates of CTB have been made available, which now allows multiple tracing with CTB. In this study, we examined the efficacy of these new AF-CTB conjugates when injected into the brain, and compared the results to our previous experiences using fluorescent 3k dextran amines. To test this, we injected AF 488 and AF 594 CTB into the anterior cingulate cortex and the medial agranular cortex in the rat, and examined the retrograde transport to the lateral posterior nucleus of the thalamus. We found that CTB was very viscous but yet very sensitive: small injection sites revealed very intense and detailed retrograde labeling. Anterograde transport was seen only when tissue at the injection site was damaged. These findings suggest that AF-CTB is a very reliable and sensitive retrograde tracer, and should be the first choice retrograde tracer for experiments examining multiple pathways within the same brain.