This study aimed at a reexamination of the presence of main cholinergic enzymes in human ejaculate. The objectives were to define the cellular location of the acetylcholine-biosynthesizing enzyme, choline acetyltransferase (ChAT) in human sperm, and to determine the levels and the source of ChAT, the acetylcholine-degrading enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in human seminal fluid. The overall goal was to provide basic understanding and support for usefulness of spermatic cholinergic system as a functional ex vivo assay for testing new potential cholinergic acting drugs. Localization of ChAT was determined with flow cytometric analysis by using different staining procedures (extracellular staining or intracellular staining) with a fluorophore conjugated anti-ChAT antibody. In addition, the activity and protein levels of ChAT, AChE and BChE were measured in 75 seminal fluid samples from different sources (epididymis, seminal vesicles, prostate), using three different colorimetric enzyme activity assays that were integrated with ELISA protein measurement assay. Human spermatozoa were found to express the acetylcholine biosynthesizing enzyme, ChAT, both intracellularly and extracellularly, although most of this enzyme was present intracellularly. The main source of these main cholinergic enzymes in seminal fluid seemed to be from spermatozoa. Overall, the findings indicate that using sperm motility as a read out for monitoring effect of compounds that target cholinergic system may be useful if the whole ejaculate rather than washed sperm is used due to presence of substantial amount of soluble forms of the enzymes in the samples.