The study described involves an attempt to identify 17 beta-fatty acid esters of testosterone in blood plasma following administration of such agents. These drugs are therapeutic but are increasingly misused by athletes in an attempt to improve sports performance. The mass spectral properties of testosterone esters under electrospray ionization are described. These esters (testosterone acetate, propionate, isocaproate, benzoate, enanthate, cypionate, phenylpropionate, decanoate, and undecanoate) essentially give only a protonated molecular ion (MH+) under "optimum sensitivity" mass spectrometric conditions but could be induced to fragment in the source or collision cell of a triple quadrupole mass spectrometer. The underivatized steroid esters were analyzed by direct infusion because development of solvent systems compatible with high-performance liquid chromatography (HPLC) was not achieved for these nonpolar compounds. HPLC/MS (mass spectrometry) was possible when the steroids were converted to polar, water soluble, Girard hydrazones, and almost all were separated by microbore C4 HPLC using a water, acetonitrile, TFA gradient. The mass spectra under optimal ionization conditions essentially comprised only a molecular ion (M+), but source fragmentation gave major ions at M - 59 and M - 87 for all compounds. The molecular ion and these fragment ions were monitored in a selected-ion-recording (SIR) method developed for detecting the steroids in plasma. Using this methodology, testosterone enanthate and undecanoate could be detected after intramuscular injection or oral administration of the drugs. Further development of the technique could form the basis of a protocol for confirming the misuse of testosterone in sport, especially if sensitivity could be improved.