OBJECTIVE: To examine the underlying factors leading to infertility in a male patient from whom phospholipase C zeta H398P (PLCzeta(H398P), histidine > proline) and PLCzeta(H233L) (histidine > leucine) mutations were previously identified. DESIGN: Laboratory-based study. SETTING: University laboratory. PATIENT(S): An infertile 38-year-old man with significantly impaired oocyte activation ability. INTERVENTION(S): Minisequencing of individual sperm for PLCzeta(H398P) and PLCzeta(H233L), and investigation of localization patterns arising from the expression of fluorescently tagged PLCzeta isoforms in HEK293T cells. MAIN OUTCOME MEASURE(S): The presence/absence of PLCzeta(H398P) and PLCzeta(H233L) determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLCzeta isoforms quantified in HEK293T cells. RESULT(S): Sperm possessed either PLCzeta(H233L) or PLCzeta(H398P), but never both at the same time. Fluorescent PLCzeta(H233L) and PLCzeta(H233L+H398P) (both mutations together) localized to discrete regions in HEK293T cytoplasm but not the plasma membrane. Fluorescence statistically significantly varied between constructs such that PLCzeta(WT) > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLCzeta(H233L+H398P) exhibited a statistically significantly reduced level of fluorescence compared with PLCzeta(H398P) at 48 hours but not 56 hours. CONCLUSION(S): Both H398P and H233L mutations are present on different alleles and do not alter PLCzeta localization in HEK293T cells. Loss-of-activity mutations in PLCzeta may contribute not only toward male infertility but also male subfertility in cases where PLCzeta is mutated on a single allele.