Nonylphenol belongs to the most investigated xenohormones acting at the estrogen receptor. Technical nonylphenol contains approximately 20 para-substituted isomers. Because of limitations in testing and quantifying all 20 isomers in the mixture, the linear form, 4n-NP, is often used as a reference substance, even though it is not present in the technical mixture. Here, we report on the synthesis and estrogenic potency of six nonylphenol isomers that occur at different proportions in technical nonylphenol mixtures. The relative potency of each isomer was determined by use of the MVLN transcriptional activation cell assay. As well, a subset of isomers was tested in the E-screen assay. One isomer, p353-NP, exhibited the same relative potency as the nonylphenol mixture, whereas the other isomers were found to be less potent. Two isomers, p22-NP and p262 NP, and the linear 4n-NP were found to be weak ER agonists with responses near the detection limit in the MVLN assay. Two isomers, p262-NP and 4n-NP, exhibited measurable activity in the E-screen. Our results demonstrate that defined p-NP isomers are most suitable for reflecting the estrogenic potency of technical NP mixtures. Among other applications, they should be used in the future to explain differences in estrogenic potency due to NPs as detected by various in vitro assays.