The ankryn repeat domain proteins, Ankrd1 and Ankrd2, are expressed at the highest levels in skeletal muscle and heart where they are localized to the I band of the sarcomere through binding to titin and myopaladin. Ankrd1 and Ankrd2 migrate from the sarcomere to the nucleus when muscle is stressed, and act as coregulators for a growing number of transcription factors. Expression of Ankrd1 is altered by castration suggesting a link to androgen action. This investigation explored the effects of testosterone on Ankrd1 and Ankrd2 expression and determined whether Ankrd1 or Ankrd2 binds to or regulates the transcriptional activity of the androgen receptor (AR). Incubation of rat L6 myoblasts expressing the human AR (L6.AR) with testosterone reduced mRNA levels for Ankrd1 by approximately 50% and increased those for Ankrd2 by 20-fold. In reporter gene assays conducted with CHO cells co-transfected with an ARE-Luc reporter gene, Ankrd1 blocked the ability of testosterone to increase reporter gene activity while Ankrd2 had no effect. The effect of Ankrd1 and Ankrd2 on repression of the MAFbx promoter by testosterone was also tested in C2C12 cells using an MAFbx-Luc reporter gene (pMAF400-Luc); Ankrd1 blocked repression of pMAF400-Luc by testosterone while Ankrd2 did not. Co-immunoprecipitation studies revealed that Ankrd1 bound to the AR whereas Ankrd2 did not. The effect of Ankrd1 or Ankrd2 on changes in gene expression induced by testosterone in L6.AR cells was also evaluated. Incubation of L6.AR cells with testosterone modestly reduced myogenin mRNA levels but did not significantly alter those for mdm2, MEF2d, TnnI1, TnnI2, or p21. When cells were transfected with Ankrd1, testosterone markedly reduced mRNA levels for MEF2d, myogenin, p21 and TnnI1, increased those for TnnI2, but did not alter those for mdm2. When cells were transfected with Ankrd2, testosterone increased MEF2d and myogenin mRNA levels, having the opposite effect to cells transfected with Ankrd1; Ankrd2 did not change the effects of testosterone on TnnI1, TnnI2, p21, or mdm2 mRNA levels. In conclusion, testosterone regulates the expression of Ankrd1 and Ankrd2; Ankrd1 binds to and directly regulates the transcriptional activity of the AR whereas Ankrd2 does not; expression levels of both Ankrd1 and Ankrd2 modulate effects of testosterone on gene expression in cultured myoblasts. Published by Elsevier Inc.