14-3-3 Proteins are a class of regulatory protein that affect the activity of a wide range of targets by direct protein–protein interaction. In plants such as Arabidopsis and tomato, 14-3-3s are encoded by a family of around 12–13 genes. With multiple 14-3-3 isoforms and many more target proteins, it is difficult to assign specific biological functions to 14-3-3 proteins. Here, we describe two different approaches that identify roles for 14-3-3 proteins in signalling and plant development. Firstly, we have shown that 14-3-3 proteins are required for pollination in tobacco, by disrupting the function of pollen 14-3-3 proteins via two different mechanisms. Firstly, we used RNAi to silence the most abundant tobacco pollen 14-3-3 gene, and secondly, we used a pollen-specific promoter to express a generic 14-3-3 inhibitor protein. Pollen with reduced 14-3-3 activity produced by both methods demonstrated a reduced ability to fertilise tobacco ovules. Secondly, we have identified the Arabidopsis ethylene signalling protein, CTR1, as a 14-3-3-interacting protein. We find that mutation of a 14-3-3 binding site in CTR1 blocks its activity when introduced into transgenic plants, resulting in constitutive ethylene signalling and aberrant plant development. These data clearly indicate a requirement for the CTR1-14-3-3 interaction for normal ethylene signalling in Arabidopsis.