▼The two-hybrid system (THS), first introduced by Fields and Song (Ref. 1), is a powerful technique for identifying new proteins involved in specific biological processes. It allows for the rapid isolation of the gene that codes for a protein that interacts with a specific protein of interest. Here, we present not a review of the current THS technology but, rather, a comprehensive guide designed to take the reader through a THS screen. Readers not familiar with the THS are asked to review (Ref. 1, 2, 3, 4, 5). Currently, there are a number of different versions of the THS available. This article describes procedures useful for the ‘Fields’ THS, based on the GAL4 transcription factor. Many of the following protocols can be used with other versions with some modification; however, they might not be directly applicable to the Brent Interaction Trap version because of some distinct differences (Ref. 6). This third article of the three on the yeast two-hybrid system describes the characterization of THS positives. Primary THS positives that activate both the HIS3 and the lacZ reporter genes can now be subjected to further analysis. Due to the in vivo nature of this system, unforeseen obstacles might be encountered that require you to return to a previous step. If less than 20 positives are obtained, follow section C1 (see Figure 1 for a schematic) through each step until all are characterized. If greater than 20 positives are identified, proceed to section C9 and use segregation analysis to eliminate false positives. Continue the analysis with remaining positives by returning to section C1. In those cases where it is difficult to isolate the AD:cDNA library plasmid responsible for reporter-gene activity (in steps