Summarize:

To ascertain the sex of a cat or kitten, it's necessary to pick the cat up. Some cats don't like to be handled, so take a little time to let him or her get comfortable around you.  Stand or crouch near the cat and let him or her approach you. When the cat comes close, let him or her sniff your hand. If the cat seems nervous, you may have to come back later or have a partner help you with the next step. Gently pick up the cat and cradle him or her in one arm. Use your free hand to lift the tail so you can examine the genital area.  If the cat doesn't mind being handled, this may be easiest to perform while sitting on a chair or the couch, so you don't have to worry about dropping the cat. If you're working with a partner, have the partner hold the cat securely in both arms while you lift the tail. If the cat avoids having his or her tail lifted, try scratching him or her in the area where the tail meets the back. Cats usually lift their tails when they are touched in this spot. The surest way to tell the difference between a male and female cat is by examining the cat's physical features below its tail. Begin by looking for male genital features, which can be somewhat easier to spot.  A male cat will have an anus, scrotum, and penis, while a female cat will have only an anus and urinary tract opening. In an entire (or tom) cat, the scrotum is covered with fur and contains two testicles, each typically ranging in size from the size of cherry pits to actual cherries. The scrotum sticks out from the male cat's rear as a fairly obvious pair of bumps. If the cat has long fur, this can make the scrotum more difficult to see, in which case try dampening the fur with water to flatten it down and make the scrotum more obvious. A neutered male cat will still have a scrotum, though it will typically be smaller.  The penis is sited below the scrotum, beneath the skin, and exits at a small furry mound between the cat's thighs. You might imagine a male cat’s genitalia resembling a colon (:). A male cat's anus and urinary tract opening are at least 1 inch (2.5 cm) apart, or 1⁄2 inch (1.3 cm) for kittens. If the cat’s genitalia doesn’t appear to match that of a male cat, begin looking for female traits.  A female cat will have an anus and a urinary tract opening/vulva, with the vulva in the shape of a vertical slit. You can imagine a female cat’s genitalia as resembling a semi-colon (;).  A female cat will have a shorter distance between anus and vulva, typically about 1⁄2 inch (1.3 cm) apart.
Approach the cat or kitten with care. Lift the cat's tail. Look for genitalia characteristic of a male cat. Look for genitalia characteristic of a female cat.