||Quantifying the larval dispersal component of population connectivity is extremely challenging due to the many difficulties associated with directly observing larvae in their marine environment. Transgenerational isotope labeling is a recent empirical technique that addresses this challenge. It relies on the transmission of an artificially enriched stable isotope (e.g., Ba-137) from gravid females to the embryonic otoliths of their offspring, allowing for mass permanent marking of larvae. Before implementing transgenerational isotope labeling in the wild, it is essential to investigate the transmission longevity of the mark from females to larvae and to assess the potential negative effects on females and their offspring. We injected females of the Humbug damselfish, Dascyllus aruanus, with an enriched Ba-137 solution and reared the resulting progeny to test the marking success and the transmission longevity of the mark, as well as determine potential effects of transgenerational isotope labeling on spawning frequency and size of 1-day eggs and 2-day larvae. Three different single-injection dosages (0.5, 1 and 5 mu g of Ba-137 g(-1) fish weight) were tested, as well as monthly repeated injections of the lowest dosage over a whole reproductive season. We implemented a new method that allows extracting otoliths of newly hatched larvae and analyzing them using laser ablation coupled plasma mass spectrometry (ICP-MS). We showed that for D. aruanus, injection with a low dose (0.5 mu g Ba-137 g(-1), fish weight) produced consistently significantly marked larvae with a half-life for successful enriched Ba mark transmission of approximately 1 month, and that monthly repeated injections of this dose did not negatively impact spawning success or condition of eggs and larvae. Monthly repeated injections of enriched Ba isotope injections at 0.5 mu g Ba-137 g(-1) fish weight will therefore present an effective means of mass marking D. aruanus larvae throughout an entire reproductive season.