The consistency of results using Nile blue staining of fetal cells in amniotic fluid to estimate fetal maturity has been demonstrated. Recently, evidence to support this method has been published from this laboratory. Several biochemical and technical factors were cited as important to the success of the method. This report deals with further testing of several Nile blue dyes (hydrochloride and sulfate) necessitated by changes in production and federal regulation of this compound. The results show Nile blue hydrochloride (HCI) to be the most superior dye regardless of color index. Buffered solutions (6.6) of Nile blue A (sulfate) improve its performance, but not to levels demonstrated by the HCI preparation. Storage at room temperature adversely affects each dye; however, the sulfate variety appears to be the most unreliable under these circumstances. For best results, it is recommended that Nile blue HCI be used and the solution (buffered or normal) be kept refrigerated.