Serotonin, N-acetyl serotonin, and 5-methoxy-N-acetyl serotonin (melatonin) were conjugated to bovine serum albumin (BSA) using formaldehyde. The molar ratio of hapten to protein was determined spectrophotometrically. Spectrophotometric data indicated that serotonin and N-acetyl serotonin but not melatonin were conjugated to bovine serum albumin. Selected hapten–protein conjugates were suspended in Freund's adjuvant and injected into rabbits. Antisera were harvested monthly and screened by double immunodiffusion. Immunodiffusion and inhibition tests indicated that antibodies raised to serotonin–BSA reacted with serotonin and 5-methoxytryptamine but failed to cross react with N-acetyl serotonin or melatonin. Inhibition tests indicated that antibodies to N-acetyl serotonin – BSA reacted with N-acetyl serotonin and cross reacted with melatonin but not with serotonin or 5-methoxytryptamine.