The negative impact of hydrosalpinx on IVF outcome is well recognized but some reports indicate that tubal infertility with hydrosalpinx is a heterogeneous entity and may have different effects on the outcome. The embryotoxic effects of hydrosalpinx fluid (HF) have been documented in mouse but not human embryos. This study examined the effects of HF on sperm motility and survival after various periods of incubation. Fifteen infertile patients with hydrosalpinx shown on ultrasound monitoring during ovarian stimulation underwent aspiration of HF after egg collection. Electrolytes, glucose and pyruvate concentrations were within the physiological ranges found in normal human tubal fluid. Sperm motility and velocities remained unchanged after 5 h of incubation with various concentrations of HF but the percentage of motile spermatozoa was significantly reduced after 24 h of incubation. Both 50 and 100% HF were potentially cytotoxic (survival indices <85%). The detrimental effect seemed to be dependent on the concentrations of HF. Low osmolarity, low lactate concentrations or the protein content may be responsible for the loss of sperm motility. A human sperm survival test using HF may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment or tubal surgery.