The content is divided into 14 chapters. The first chapter serves as an introduction and considers the mythological and geological origin of night. The next 12 chapters are each titled after an hour starting with chapter 2's 6 pm and ending with chapter 13's 5 am. Chapter 2 discusses the transition into night, including sunsets, the green flash, the stages of twilight, as well as Olbers' paradox, and a definition of the size and speed of night. The 7 pm chapter deals with nature at night, how animals see and hear differently at night with a focus on bats, nighthawks, and nocturnal insects. The 8 pm chapter analyzes children's literature and bedtime stories. The 9 pm chapter discusses aspects of the city at night, including the evolution of nightclubs, street lights, and the impacts of light pollution. The 10 pm chapter discusses night festivals around the world and throughout history. The 11 pm chapter explains the circadian rhythm and the physiology of sleep and dreams.The attacks would almost always strike during the REM phase of the victim's first sleep cycle of the night... [One patient] who had survived several attacks said that, during each attack, something came into his bedroom, sat on his chest, and tried to crush the air out of his lungs. In one nightmare it was a cat, in another a dog, and once it was a woman. Dewdney on sudden unexplained death syndrome, Acquainted with the Night, pages 177–178.
The 12 am chapter traces the history behind dream interpretation from Gilgamesh to Sigmund Freud, Carl Jung, and Calvin Hall. Here, Dewdney considers nightmares and takes sudden unexplained death syndrome, whose sufferers are almost always Asian males, between 20 and 49 years old, as an extreme example of nightmares that cause the dreamer to die from a "ventricular fibrillation...brought about by extreme terror". The 1 am chapter compares literary and mythological personifications of, or beings associated with, the night. The 2 am chapter tells the stories of the legends behind the moon and the constellations. The 3 am chapter is all about insomnia. The 4 am chapter provides a geographical aspect, touring the places with long nights, like Las Vegas, caves, the poles, and deep within the oceans. The 5 am chapter discusses artistic representations of night, especially in music, on film and through paintings. The final chapter is a conclusion in which Dewdney reflects on memorable sunrises he has experienced and contrasts sunrises with sunsets.